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Latest revision as of 02:19, 27 October 2010

notebook

Week 1	6-14-2010	06-15-2010	06-16-2010	06-17-2010	06-18-2010
Week 2	06-21-2010	06-22-2010	06-23-2010	06-24-2010	-
Week 3	06-28-2010	06-29-2010	06-30-2010	07-01-2010	07-02-2010
Week 4	-	07-06-2010	07-07-2010	07-08-2010	07-09-2010
Week 5	07-12-2010	07-13-2010	07-14-2010	07-15-2010	07-16-2010
Week 6	-	-	07-21-2010	07-22-2010	07-23-2010
Week 7	-	07-27-2010	07-28-2010	07-29-2010	07-30-2010
Week 8	08-02-2010	08-03-2010	08-04-2010	08-05-2010	08-06-2010
Week 9	08-09-2010	08-10-2010	08-11-2010	08-12-2010	08-13-2010

06-14-2010 [top]

LacI Transformation

Performed bacterial transformation according to [http://openwetware.org/wiki/Silver:_Bacterial_Transformation Silver Lab Bacterial Transformation], however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.

Transformed the following, each into its own tube of TOP10 E.Coli:

1) LacI with a rapid degredation tail attached, biobrick BBa_C0012, located on plate 1, well 2O, plasmid pSB1A2

2) LacI wildtype, biobrick BBa_I732100, located on plate 2, well 10E, plasmid pSB1A3

Streaked 100μL each of 1 and 2 onto separate LB + Amp dishes, along with a control dish of 100μL untransformed TOP10 E.Coli cells.

Plates incubated overnight, colonies observed in all plates except the control.

[click to enlarge]

06-15-2010 [top]

Oligonucleotides for LacI
the following sequences for LacI and a Nuclear Localization Signal (NLS) were ordered for PCR:

LacIn.BB.Rev

   5'-AAG GCT GCA GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA AGA CTG CCC GCT TTC CAG TCG GGA AA - 3'

LacIn.BB.Fwd

   5'- CCT TGA ATT CGC GGC CGC ATC TAG AAT GAA ACC AGT ACC GTT ATA CGA TGT C -3'

NLS.BB.Rev

   5'-GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTTCTT AGG ATG AAC AAC AGA AGA -3'

NLS.BB.Fwd

   5'-CTA GAT CTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA -3'

VP16 transcription activating domain bacterial transformation

Performed bacterial transformation according to Silver:_Bacterial_Transformation, however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.

Transformed the following into its own tube of TOP10 E.Coli:

VP16 Eukaryotic Activating Domain, biobrick BBa_K105001, located on plate 3, well 13C, plasmid pSB1A2.

In the course of reconsitituting the the biobrick plasmid from its well, too little DH₂O was added (1μL), so an additional 10μL were added to the well.

The first tube of TOP10+biobrick plasmid was erroneously heatshocked before being chilled for 30 mins on ice, so a second tube of TOP10 cells was introduced, and the correct proceedure followed for the second tube. Additionally, the first tube was then allowed to chill and heat shock again, ultimately producing two tubes of transformed TOP10 cells.

100μL of both tubes were streaked onto an LB + Amp dish, and incubated at 37°C on the plates overnight. Colonies the following morning, colonies were observed on both plates, although fewer on the plate containing the E.Coli which were erroneously heat-shocked too soon.

LacI Miniprep Preparations
Prepared 6 overnight cell cultures, consisting of three colonies each from the LacI wt and LacI+rapid degradation tail E.Coli cells transformed previously.

Each culture consisted of 5mL of LB+Amp, and a scraping of 1 E.Coli colony, in a 100cc tube.

These overnight cell cultures were set to shake at 37°C overnight.

06-16-2010 [top]

LacI Miniprep

Performed miniprep of overnight cell cultures of 3 colonies each of wildtype LacI transformed bacteria and LacI+rapid degradation tail bacteria.

Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].

LacI Nanodrop Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

Plasmid	Quantity (ng/μL)	260/280
O2 #1	77.1	1.81
O2 #2	90.1	1.9
O2 #3	50.4	1.95
E10 #1	99.6	1.93
E10 #2	104.6	1.92
E10 #3	58.6	1.98

LacI Plasmid Digest
We digested 15μL of each of the 6 samples with 1μLH₂O, 1μL EcoR1, and 1μL Pst1 (high efficiency restriction enzymes).

Ran an E-gel of the digest results.

Lane #	1	2	3	4	5	6	7	8	9	10
O2 #1		ladder	O2 #1	O2 #2	O2 #3		E10 #1	E10 #2	E10 #3

[click to enlarge]

VP16 Colony Inoculation
Prepared 3 10cc tubes each containing .5μL LB+amp and cells from one of the three colonies of the VP16 cell culture of the previous night (colonies from the second-tube plate, the cells in which were heatshocked in the correct order).

Tubes were set to shake at 37°C overnight.

Preparing Glycerol Stocks of LacI wt and LacI+LVA
Prepared 2 cryo tubes, with the following labeling:

Tube 1) contains BBa_C0012, from plate 1, well 2O

On top: '#1'

On side: '6-16-10 MP pSB1A2 LacI+LVA'

Tube 2) contains BBa_I732100, from plate 2, well 10E

On top: '#2'

On side: '6-16-10 MP pSB1A3 LacI Wildtype'

Each cryo tube contains .5mL of the respective overnight cell culture (O2 colony #1 and E10 colony #1), and .mL of 80% glycerol.

Both Cryo tubes were placed in the box labeled 'Genetic Fence iGEM 2010' in the -80°C freezer.

06-17-2010 [top]

VP16 Miniprep

Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:

VP16

Protocol

4ml from each overnight cell culture of VP16 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm
remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time
excess LP+amp decanted, the pellet resuspended in 250μL P1
contents transferred from each 15ml conical to a new epindorf tube
250 μL P2 added to each tube, mixed by inverting gently 4-6 times
350 μL N3 added to each tube, mixed by inverting as before
tubes centrifuged for 10 minutes at 13,000 rpm
supernatants pipetted into new QIAprep sin columns
columns centrifuged for 30-60 seconds, flow through discarded
QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds
columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds
flow through was discarded and tubes centrifuged for an additional minute
column was placed in a clean 1.5 ml microcentrifuge tube
50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after
flow through kept and purity tested

Nanodrop Values for VP16

VP16 #1
- 95.7 ng/μL
- 260/280 = 1.92

VP16 #2
- 102.4 nm/μL
- 260/280 = 1.92

VP16 #3
- 90.7 ng/μL
- 260/280 = 1.92

Digestion

Quantities per double digestion reaction

10μL sample
1μL EcoRI
1μL PSD1
2μL buffer
6μL H20

Single digest reaction

10μL sample
1μL EcoRI
2μL buffer
7μL H20

ingredients combined and all tubes were put in 37°C bath for 10 minutes

Digest with SPE1 and XBA1

10μL sample
1μL SPE1
1μL XBA1
2μL buffer
6μL H20

All 3 epindorfs of VP16 were combined for a total volume of 90 μL or 9μg
1 unit enzyme cuts 1 μg DNA in 30-60 minutes

Digestion Gel

Ingredients

digested enzymes from above reaction
1 eppendorf containing 2μL uncut VP16 and 18μL H2O
kb ladder
IMAGE TO BE ADDED + details

Gel Recipe: 1 X TAE

900 ml dH2O
100 ml TAE

1 gel

150 ml buffer TAE
add ~2.8g agarose
mix in beaker
microwave until completely dissolved
add 5 μL ethedium bromide per 100 ml gel after solution has cooled (no longer steaming)
pour gel and let sit til solidified

Procedure

run products on gel for ~45 minutes
cut out band containing DNA fragment
follow extraction protocol in QIAquick spin handbook for QIAquick gel extraction kit protocol

Nanodrop values for resulting DNA

5.6ng/μL
260/280 = 2.24

Tube placed in -20 freezer labeled VP1 fragment XBA SPE1

Glycerol Stocks of VP16

0.5 ml 80% glycerol stock combined with 0.5 ml overnight culture of colony #2 of V{16 TP{10 cells
final product placed in the team fence box after mixing in the -80°C freezer

Bacterial transformation of GAL4 DNA binding domain

BBa_K105007, plate 3 well 9I, psB1A2

Procedure

removed 1 tube TOP10 chemically competent E.Coli and placed on ice
cleaned biobrick depository w/ ethanol
pipetted 1 μL of plasmid into the TOP10 cell tube
placed TOP10 cell tube on ice for 30 minutes
heat shocked at 42°C for 30 seconds
placed on ice for 2 minutes
added 170 μL SOC medium
streaked on LB + amp paltes, incubated at 37°C overnight

06-18-2010 [top]

LacI+NLS PCR

LacI+NLS primers arrived in mail

spun at 14,000 rpm for 10 minutes diluted stocks to 100 μM

   LacIn.BB.Rev (1.7nM) diluted with 17μL DH₂O
   LacIn.BB.Fwd (1.5nM) diluted with 1011μL DH₂O
   NLS.BB.Rev (4.2nM) diluted with 42μL DH₂O
   LacIn.BB.Fwd (101.1nM) diluted with 15μL DH₂O

PCR recipe

20μL per reaction
1μL template plasmid
1μL R primer
1μL F primer
4μL HF buffer 5X
2μL DNTPs
0.5μL pFU polymerase
rest H2O to make the volume per tube 20μL

used LACIN program in phusion PCR machine to perform PCR

06-21-2010 [top]

Barnase and Barstar

Barnase and Barstar Plasmids from ADDGENE arrived in mail

pMT316 - Barstar. [http://www.addgene.org/pgvec1?f=c&identifier=8608&atqx=pMT316&cmd=findpl Info from ADDGENE]

pMT413 - Barnase, Barstar. [http://www.addgene.org/pgvec1?f=c&identifier=8606&atqx=pMT413&cmd=findpl Info from ADDGENE]

pMT1002 - Barnase, Barstar. [http://www.addgene.org/pgvec1?f=c&identifier=8621&atqx=pMT413&cmd=findpl Info from ADDGENE]

Notes from lab meeting

Karmella suggested we try adding an LVA tail to the Cre we use for the crelox fence system.

GAL4 DBD Innoculation

Pipetted 5mL of LB+Amp each into capped cc tubes, and mixed bacterial scrapings from 5 separate colonies of transformed GAL4 DNA binding domain E.Coli. Set to shake at 37 degrees C overnight.

06-22-2010 [top]

GAL4DBD Miniprep

[click to enlarge]

HindIII + PstI digest of miniprepped V9-V12 (#5,6) to check whether insert is present.]

pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute
remaining overnight cell cultures were placed in fridge
decanted LB+amp, resuspended cells in 250 μL P1 buffer
contents transferred to eppendorfs
250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times
350 μL N3 buffer added to each, tubes inverted 4-6 times
centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns
centrifuged for 30-60 seconds, flow through discarded
0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute
QIAprep columns put into new eppendorfs
50 μL buffer EB added to columns, let stand for 1 minute
centrifuged for 1 minute at 13,000 rpm
tubes labeled "GAL4" plus the specific colony number

PCR

PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)

1=95°C for 10:00

2=95°C for 00:15

3=50°C for 00:30

4=72°C for 01:30

5=steps 2-4 X 29

6=72°C for 10:00

7=4°C for ∞

[click to enlarge]

PCR successful

PCR for E10 LacI

above PCR was repeated without O2 so as to finalize our E10 biobrick for use
PCR was performed using the standard proportions of reagents
The above settings on the PCR machine were used

[click to enlarge]

Gel cut-out

To Make Agarose Gel

150 mL TAE buffer combined with 1.5 g agarose in beaker
solution microwaved until agarose disolved
7.5 μL Ethidium Bromide added after beaker was no longer steaming
solution poured into clean gel mold and gel was left to solidify
E10 PCR product from the morning was consolidated into one tube,
final volume =90μL PCR product + 18μL 5X loading dye =110μL

DNA was extracted from gel by slicing out the bands and DNA was purified using the QIAquick Gel Extraction Kit, following protocol specific to using a centrifuge

see [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2 QIAquick spin handbook]

Innoculations

Colonies from the following cultures were innoculated and set to shake at 37°C overnight:

PMT413
PMT316
firefly luciferase
GFP (mut)
cre recombinase
lox66
lox71

06-23-2010 [top]

Minipreps
The following colonies were minipreped after passing the night in the shaking incubator (37°C)

PMT413
PMT316
firefly luciferase
GFP (mut)
cre recombinase
lox66
lox71

Nanodrops for above minipreps

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

If two numbers are given, separated by a slash, then two measurements were taken, both are presented.

Plasmid	Quantity (ng/μL)	260/280
Lox 71 #1	41.4	1.82
Lox 71 #2	59.6	1.89
Lox 71 #3	17.4/80.2	1.98
GFP #1	21.2	1.83
GFP #2	110.0	1.90
GFP #3	60.9/124	1.88
pMT316 #1	11.6	1.76
pMT316 #3	16.5	1.81
Cre #1	84.9	1.93
Cre #2	107.7	1.88
Lox66 #2	51.3	1.87
Firefly Luciferase #2	309.3	1.91
Firefly Luciferase #3	325	1.9
pMT413 #1	461.7	1.89
pMT413 #2	362.2	1.91
pMT413 #3	381.8	1.92

06-24-2010 [top]

Primers for Barstar, Barnase, and Gal4DBD arrive Primers:

BB.Barstar.Fwd - 36.1 nM, diluted with 361μL H₂O

  5'- CCT TTC TAG AAT GAA AAA AGC AGT CAT TAA C -3'

BB.Barstar.Rev - 30.0 nM, diluted with 300μL H₂O

  5'- AAG GCT GCA GCGGCC GCT ACT AGT TTA AGA AAG TAT GAT GGT GAT GTC -3'

BB.Barnase.Fwd - 38.5 nM, diluted with 385μL H₂O

  5'- CCT TTC TAG AAT GAA AAA ACG ATT ATC ATG G -3'

BB.Barnase.Rev - 13.2 nM, diluted with 132μL H₂O

  5'- AAG GCT GCA GCG GCC GCT ACT AGT TTA TCT GAT TTT TGT AAA GGT CTG -3'

BB.Gal4DBD.Fwd - 38.5 nM, diluted with 385μL H₂O

  5'- CCT TTC TAG AAT GAA GCT ACT GTC TTC TAT -3'

BB.Gal4DBD.Rev - 32.9 nM diluted with 329μL H₂O

  5'- AAG GCT GCA GCG GCC GCT ACT AGT CGA TAC AGT CAA CTG TCT TTG -3'

Digestion

Our first digestion was unsuccessful except for the confirmation of the PMT316 fragment; this can be attributed to an incorrect combination of restriction enzymes. The digestion was performed again in the afternoon using EcoRI + SpeI for the biobricked plasmids (lox71, GFP mut, lox66, cre recombinase, and firefly luciferase) and EcoRI + HindIII for PMT413.

Gel Image, first digestion:

[click to enlarge]

PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)

[click to enlarge]

The length of the shortest bands were confirmed to be roughly the same length of the desired excerpt with the exception of Lox66 and Lox71 where it was determined that the inserts being only 34 bp had run off the gel, therefore the single band seen was the remaining plasmid.

Yeast Growth Assay To assay the effect of Methoxyfenozide on yeast, three flasks containing 25μL DMSO (control), 2.5μL of 100mM Methoxyfenozide stock in 25mL DMSO to form the 1μM condition, and 25μL of 100mM Methoxyfenozide stock in 25mL DMSO to from the 10μM condition. Each beaker also contained y190 yeast strains and YPD medium.

To construct a growth curve of the yeast under the 0μM, 1μM and 10μM conditions, 1mL from each flask would be pipetted into a cuvette and measured in a spectrophotometer ever hour to assess yeast density.

Unfortunately in this first attempt, no growth was observed after 4 hours, in the control and experimental groups, so the test was called off, to be repeated tomorrow.

Time	0μM Methoxyfenozide	1μM Methoxyfenozide	10μM Methoxyfenozide
11:00 AM	.045	.042	.036
12:00 PM	.036	.040	.041
3:00 PM	.035	.036	.039

06-28-2010 [top]

B21 Innoculation Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.

PCR of Gal4DBD and Barnase, attempt 2 Ran 3 tubes of each for a total of 6. 7x Mastermix:

14μL DNTP
3.5μL Polymerase
28μL 5x Buffer
73.5μL DH₂O

Each tube contained:

1μL Fwd primer
1μL Rev Primer
1μL Minipreped sample
17μL Mastermix

Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program

Gel of Gal4 DBD and Barnase from second PCR attempt Ran 1.2% E-gel of the 6 PCR product tubes.

1	2	3	4	5	6	7	8	9	10	11	12
	Ladder		Gal4 DBD 1	Gal4 DBD 2	Gal4 DBD 3	Barnase 1	Barnase 2	Barnase 3		Ladder

[click to enlarge]

QIAQuick Purification of PCR Product
Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.

as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)
pipetted the mix into a QIAquick spin column, spun for 30 seconds at 13,000 rpm
discarded flow-through
added .75mL PE buffer to the column, spun for 30 seconds
discarded flow-through, and spun again for 1 minute
moved the column to a fresh eppendorf tube
added 50 μL EB buffer to the column, spun for 1 minute
labeled the eppendorf tube 'Barstar PCR purified'

06-29-2010 [top]

B21 plasmid maxiprep

colony inoculated overnight and medium was centrifuged to obtain a pellet.

followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition)

06-30-2010 [top]

pMT1002 Miniprep

4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm
remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time
excess LP+amp decanted, the pellet resuspended in 250μL P1
contents transferred from each 15ml conical to a new epindorf tube
250 μL P2 added to each tube, mixed by inverting gently 4-6 times
350 μL N3 added to each tube, mixed by inverting as before
tubes centrifuged for 10 minutes at 13,000 rpm
supernatants pipetted into new QIAprep sin columns
columns centrifuged for 30-60 seconds, flow through discarded
QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds
columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds
flow through was discarded and tubes centrifuged for an additional minute
column was placed in a clean 1.5 ml microcentrifuge tube
50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after
flow through kept, eppendorfs labeled pMT1002 plus their colony number

Also see pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook] for miniprep instructions. pMT1002 Miniprep Nanodrop values

Plasmid	Quantity (ng/μL)	260/280
pMT1002 #1	68.2	1.84
pMT1002 #2	138.7	1.84
pMT1002 #3	127.2	1.88

PCR of miniprepped Barnase

20μL per tube

In each tube:

1μL Forward primer
1μL Reverse primer
0.5μL polymerase
4μL 5X buffer
2μL DNTPs
1μL DNA sample
10.5μL water

3 samples were made for the Barnase

B21 Plasmid Digest put 10x green FD buffer on ice to thaw.

Tube A:

1μL Xba1

1μL Pst1

2μL green 10x FD buffer

3.4μL colony A plasmid (the amount used to get .5μg of plasmid)

12.6μL H₂O (to bring the total to 20μL)

Tube B:

1μL Xba1

1μL Pst1

2μL green 10x FD buffer

1.7μL colony B plasmid (the amount used to get .5μg of plasmid)

14.3μL H₂O (to bring the total to 20μL)

Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut

[click to enlarge]

Second Attempt

Tube A:

1μL Xba1

1μL Pst1

5μL green 10x FD buffer

12.5μL colony A plasmid (the amount used to get .5μg of plasmid)

30.5μL H₂O (to bring the total to 50μL)

Tube B:

1μL Xba1

1μL Pst1

5μL green 10x FD buffer

6.6μL colony B plasmid (the amount used to get .5μg of plasmid)

36.4μL H₂O (to bring the total to 50μL)

Ran on a 1% agarose gel, 100 Volts for 30-40 mins.

Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A

[click to enlarge]

Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.

Other

Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.
Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.

07-01-2010 [top]

B21 Backbone Gel Extraction

followed QIAquick gel extraction handbook except melting temperature was 42°C instead of 50°C
4 gels to begin with, highest nanodrop value before consolidation = 1.4ng/μL
The 4 tubes were consolidated into 2 and placed in the vacufuge for ≈30 minutes to increase DNA concentration
The 2 tubes were consolidated into 1 for a final concentration of 18.6ng/μL

PMT1002 Digestion

2 reactions each containing the following:
- 1μL EcoRI
- 1μL HindIII
- 2μL 10X digestion buffer
- 1/2 ng of sample ≈7μL
- 9μL dH2O

only one band was visible in the gel for each reaction. Julia forgot to put the ladder in so it was not possible to precisely determine the single band size, but judging from the position of the loading dye it seemed that the plasmid had only been cut once.

Ligation

Ligation performed on Barstar, NLS, and LacIN

3 times as much insert as vector per reaction, length of insert taken into consideration.
- Barstar: 0.4 kb = 18.5ng
- NLS: 0.06 kb = 3ng
- LacIN: 1.1 kb = 52 ng

Ligation Recipe:

10μL insert + backbone
10μL ligation buffer (vortexed thoroughly)
1μL ligase, mix gently

let stand for 15 minutes at room temperature, then put on ice use for 4μL of the product for transformation and freeze the rest

LacIN

3μL B21 vector
7μL LacIN insert

NLS

3μL B21 vector
1μL NLS insert
6μL dH20

Barstar

3μL B21 vector
1.7μL Barstar
5.3μL dH20

Transformation from ligation

thawed 4 chemically competent TOP10 cell tubes on ice
added 4μL of each ligase rxn to each incubated on ice for 30 minutes
heat shock at 42°C for 45 seconds
put on ice for 2 minutes
added 170μL SOC medium to each tube set to shake at 37°C for 30 minutes
pippetted each tube onto its own LB+amp plate, streak with beads
incubate at 37°C overnight

07-02-2010 [top]

pMT1002, pMT413 Digest

Digested with EcoR1 and HindIII

Lane 2 KB ladder

Lane 4 pMT1002

Lane 6 pMT413

[click to enlarge]

Barnase PCR

Standard PCR measurements were used except that a new 50X dNTP mix was used in place of the 5X buffer used in all previous PCR reactions. The amount of dH20 was adjusted for the new quantity of dNTP mix accordingly.

[click to enlarge]

07-06-2010 [top]

Inoculated 7 Barstar and 3 NLS colonies from Thursday's ligation.

Performed PCR on LacIn (E10) following standard PCR measurements as previously described.

Digestion of B21 backbone using Xba1 and Pst1

Total volume per reaction: 50 μL

2μL Xba1
2μL Pst1
10μL B21 plasmid (0.3μg/μL X 10μL = 3μg DNA)
5μL 10X digestion buffer
31μL H2O

[click to enlarge]

Digestion successful, larger bands removed for gel extraction.

Gel extraction performed according to QIAgen gel extraction protocol.

07-07-2010 [top]

Miniprep of Barastar and NLS Ligation products

Performed miniprep of overnight cell cultures of 7 colonies of Barstar, 3 colonies of NLS.

Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].

[click to enlarge]

Digestion of Barstar and NLS Ligation products

7 reactions of Barstar and 3 reactions of NLS

In each reaction:

1μL Xba1
1μL Pst1
2.5μL of sample, Barstar or NLS Plasmid, respectively, obtained from miniprep (above)
2μL Buffer
13.5μL DH₂O

Ran on a 2% agarose E-gel

Lane 1: Ladder

Lane 2-8: Barstar

Lane 9-11: NLS

[click to enlarge]

PCR of LacIN 3 reactions, each with:

1μL Fwd LacIN Primer
1μL Rev LacIN Primer
.5μL Polymerase
2μL DNTP
4μL Buffer
1μL E10 Plasmid (LacI)
10.5μL DH₂O

Using program:

1= 95°C for 10:10

2= 95°C for :15

3= 50°C for :30

4= 72°C for 1:30

5= goto 2, 29 times

6=72°C for 10:10

7=4°C forever

Gel Image of LacIN PCR result:

PCR of Barnase and Gal4DBD

5 reactions each of Barnase from pMT413, Barnase from pMT1002, and Gal4

In pMT413 Barnase reactions:

1μL Barnase2.Fwd Primer
1μL Rev.Barnase Primer
.5μL Polymerase
2μL DNTP
4μL Buffer
2μL pMT413 plasmid
10.5μL DH₂O

In pMT1002 Barnase Reactions:

1μL Barnase2.Fwd Primer
1μL Rev.Barnase Primer
.5μL Polymerase
2μL DNTP
4μL Buffer
2μL pMT1002 plasmid
10.5μL DH₂O

In Gal4DBD reactions:

1μL Gal4.Fwd Primer
1μL Gal4.Rev Primer
.5μL Polymerase
2μL DNTP
4μL Buffer
2μL Gal4DBD plasmid
10.5μL DH₂O

07-08-2010 [top]

[click to enlarge]

After receiving the new primers we performed PCR on barnase vectors PMT413 and PMT1002, LacIN, as well as the GAL4 DNA binding domain. The ladder is in lane 2, the GAL4 PCR products are in lanes 5 through 9, the barnase PMT413 lanes 10 through 14, and the LacIN PCR product is in the last 3 lanes. After PCR was confirmed successful, we decided to gel purify the LacIN because of the multiple band sizes seen in the gel. Below is the gel where LacIN was gel extracted, the chunk missing from the gel is where LacIN was identified to be.

[click to enlarge]

Gel extraction was performed according to the QIAgen gel extraction kit protocol using a microcentrifuge.

Barstar Sequencing Submited plasmids from each of the 7 Barstar colonies for sequencing.

07-09-2010 [top]

Digest of yesterday's ligations LacIN - 65μL

 1μL Xba1 
 1μL Pst1
 6.5μL FD buffer
 1μL DPN1
 50μL LacIN
 6.5μL DH₂O

Gal4DBD - 50μL

 1μL Xba1
 1μL Pst1
 7μL Gal4
 1μL DPN1
 5μL FD Buffer
 36μL DH₂O

Barnase - 50μL

 1μL Xba1
 1μL Pst1
 15μL Barnase
 1μL DPN1
 5μL FD buffer
 28μL DH₂O

Put in 37°C bath for 30 mins

Following proceedure for PCR cleanup to clean up the digestion results

added 5 volumes PB buffer to 1 volume digestion product
325μL for LacIN, 250μL for Gal4DBD and Barnase
pippetted each into a QIAquick column, spun for 30 secs
discarded flow through, put columns back in same tube, added 750μL PE buffer
spun for 30 secs, discared flow through
Spun again for 1 min
placed column in new eppendorf
added 30μL EB buffer to elute, spun for 1 min

Eppendorfs labeled "LacIN digest cleanup 7/9," "Gal4 digest cleanup 7/9," and "Barnase digest cleanup 7/9"

7-10-10 (Saturday)

Innoculating colonies from Friday's ligations

Barnase, Gal4, LacIN
4 cc tubes each, 5mL LB+amp, set to shake at 37°C overnight

7-11-10 (Sunday)

Miniprep of Barnase, Gal4 and LacIN ligations

No cells found in Barnase colonies 1 and 2 --> innoculations 3 and 4 renamed Barnase 1 and 2, replacing the cell-free cultures.

pipetted 4 ml from each overnight cell culture (excepting the cell-free Barnase cultures) into a 15 ml conical *centrifuged at 4400 rpm for 6 minute
remaining overnight cell cultures were placed in fridge
decanted LB+amp, resuspended cells in 250 μL P1 buffer
contents transferred to eppendorfs
250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times
350 μL N3 buffer added to each, tubes inverted 4-6 times
centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns
centrifuged for 30-60 seconds, flow through discarded
0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute
QIAprep columns put into new eppendorfs
50 μL buffer EB added to columns, let stand for 1 minute
centrifuged for 1 minute at 13,000 rpm

07-12-2010 [top]

Nanodrop of Barnase, Gal4 and LacIN Minipreps

Plasmid	Quantity (ng/μL)	260/280
Barnase 1	226.1
Barnase 2	336.3	1.94
Gal4 1	298.0	1.88
Gal4 2	363.3	1.92
Gal4 3	301.9	1.89
LacIN 1	376.7	1.89
LacIN 2	237.8	1.90
LacIN 3	339.4	1.92
LacIN 4	368.9	1.92

Digestion of Ligation product minipreps Xba1 and Pst1

Mastermix (10x)

10μL Xba1
10μL Pst1
20μL Buffer

Barnase 1

  4μL Barnase 1
  12μL DH₂O
  4μL Mastermix

Barnase 2

  3μL Barnase 2
  13μL DH₂O
  4μL Mastermix

Gal 1

  3μL Gal 1
  13μL DH₂O
  4μL Mastermix

Gal 2

  2.5μL Gal 2
  13.5μL DH₂O
  4μL Mastermix

Gal 3

  3μL Gal 3
  13μL DH₂O
  4μL Mastermix

LacIN 1

  2.5μL LacIN 1
  13.5μL DH₂O
  4μL Mastermix

LacIN 2

  4μL LacIN 2
  12μL DH₂O
  4μL Mastermix

LacIN 3

  2.5μL LacIN 1
  13.5μL DH₂O
  4μL Mastermix

LacIN 4

  2.5μL LacIN 1
  13.5μL DH₂O
  4μL Mastermix

Put tubes in 37°C heat bath for 30 mins, then put them on ice.

Gel of Digestions:

[click to enlarge]

Sequencing

submitted Barnase 1 and 2, Gal4 1,2, and 3, and LacIN 1,2,3, and 4 for sequencing

07-13-2010 [top]

[click to enlarge]

The yeast backbones containing the PIF3 and PHYB inserts was isolated and purified. The latter image shows where the appropriately sized bands were excised.

Nanodrop values of final product are:

PHYB 11ng/μL PIF3 20ng/μL

07-14-2010 [top]

Phenol Chloroform DNA Extraction

resuspend DNA in approximately 200μL molecular grade pure water
add 1 volume phenol chloroform
- when pipetting the phenol chloroform be sure to pipette from closer to the middle level of the liquid as the top two centimeters do not contain what we want
centrifuge at top speed for 5 minutes
collect the aqueous top layer and transfer to a clean microcentrifuge tube
- the remaining liquid is toxic and must be carefully disposed of in the "toxic waste" bottle in the fume hood
add 1 volume phenol chloroform
centrifuge at top speed for 5 minutes
collect the aqueous top layer and put into a fresh tube
add three volumes 100% ethanol and 1/10 volume sodium acetate
allow solution to incubate on ice for 15 minutes
centrifuge at top speed for 10 minutes
pipette off the aqueous solution leaving a pellet of DNA in the bottom of the tube
wash in 500μL 70% ethanol
mix tube by inverting
centrifuge again for 10 minutes at top speed
air dry pellet by leaving tube open on bench (not on ice) til dry
after pellet has dried resuspend DNA in desired volume of H2O or buffer EB
- presence of DNA can be confirmed by running a small sample on a gel

NLS Ligations Ligation of NLS.Serine

2.5μL Backbone
1.5μL of 1/100 dilution (1.5ng NLS.Serine)
6μL H₂O
10μL 2x quick ligase buffer
1μL quick ligase

B21 control

2.5μL B21 Backbone
7.5μL DH₂O
10μL 2x Quick Ligase Buffer
1μL Quick Ligase

Transformation

1μL NLS lig into TOP10 cells
Chilled on ice for 30 mins
Heat shocked at 42°C for 30 seconds
Put on ice for 2 mins
Added 170μL SOC medium
Set to shake in incubator for 30 mins
Streaked on LB+amp plates, incubated overnight

Yeast backbone gel purified nanodrops

Sample	Concentraion in ng/μL	260/280
31 (1)	8.5	3.00
31(2)	10.2	2.07
32(3)	11.1	2.31
52(4)	28.5	1.83

07-15-2010 [top]

Our previous attempt resulted in DNA with the following nanodrop values:

12.1ng/μL 260/280: 0.9
39.1ng/μL 260/280: 1.11

It was believed that the other genomic DNA provided by the Matthews Lab before had similar values with regards to purity/contamination (also approaching 1) Both team allergy and fence performed PCR multiple times unsuccessfully with these DNA samples. It was thought that the low purity may be inhibiting the PCR.

Today we used the QIAgen DNeasy Plant Mini Kit. Liquid nitrogen was poured over the arabidopsis leaves in a mortar while they were ground with the pestle. This produced a fine green powder of arabidopsis leaves from which genomic DNA was extracted starting with step seven of the QIAgen protocol following liquid nitrogen crushing. The only change made to the protocol was that 40 μL of RNase 1 was added per reaction instead of the recommended 4 μL because the concentration of our supply was ten-fold lower.

Nanodrop values were as follows:

6.7 ng/μL 260/280: 1.42
5 ng/μL 260/280: 1.48

07-16-2010 [top]

PCR of Arabidopsis Genomic DNA, attempt 2 PCR using the newly extracted DNA was unsuccessful.

The degradation signal from the Austrians was successfully inoculated and colonies were found on the agar plates. Individual colonies from the plates were inoculated into tubes of 5 ml LB+Amp and placed in the shaking incubator overnight. (37°C)

Making Glyerol Stocks Combined .5 mL 80% glycerol with .5mL overnight cell culture of: Barnase 3 (number changed to Barnase 1 from miniprep onward), LacIN1, and Gal2.

7-17-10 (Saturday)

5xGal4 Promoter Annealing Product Ligation

Made a 1 in 1000 dilution of the annealing reaction

*Nanodrop*
	ng/μL	260/280
5xGalpt 1/1000 dilution	17.6	2.25

For a ligation reaction, we want:

50ng of backbone
A 1:3 ratio of backbone:insert
3(ng of backbone / basepairs of backbone) = (ng of insert / basepairs of insert)

5xGalpt is 180 bases long, B21 backbone is 3.2kb. So we want 8.4375 ng of plasmid

To reach 8.4ng of 5xGalpt, made a 1 in 2000 dilution of the 5xGalpt Annealing product

Ligation Reaction:

1μL 1/2000 5xGalpt
2.5μL B21 Backbone
6.5μL DH₂O
1μL Quick Ligase
10μL 2x Quick Ligase Buffer

B21 Control Ligation:

2.5μL B21 Backbone
7.5μL DH₂O
1μL Quick Ligase
10μL 2x Quick Ligase Buffer

Incubated at room temp for 15 mins, put on ice and immediately transformed.

Transformations of 5xGalpt, NLS.Serine anneal, and B21 Backbone control

Thawed 3 Turbo chemically competent cells on ice
Pippeted 1μL of the ligation or annealing reaction into its own tube of turbo cells
Chilled on ice for 30 mins
Heatshocked in 42°C water on heat block for 30 seconds
Chilled on ice for 2 mins
Added 170μL SOC medium to each tube, set to shake at 37°C for 15 mins
Streaked on LB + amp plates, and left in incubator overnight

7-18-10 (Sunday)

Re-doing 5xGalpt and NLS.Serine Annealing Reactions Set ~400ml of water to boil in a beaker on a heat block.

Phosphorylating 5xGalpt

3μL 100μM oligo1.5xgal4
3μL 100μM oligo2.5xgal4
3μL 100μM oligo3.5xgal4
3μL 100μM oligo4.5xgal4
3μL 10x PNK buffer
2μL nM ATP
2μL t4 PNK
11μL DH₂O

put in 37°C waterbath for 90 mins Then added 4μL 5M NaCl to halt the reaction

To get rid of the NaCL so that I can ligate the overlapping oligos:

added 3 volues of 100% ethanol (90μL), spun at top speed for 20 mins
pippetted off and discarded the supernatant, added 100μL of 70% ethanol, broke up the pellet, and spun for 5 mins
pippeted off supernatant, allowed to air dry.
added 2μL T4 ligase buffer, 1μL T4 ligase, and 17μL DH₂O, using this mix to resuspend the pellet
set in PCR machine at 16°C for 12 hours, then 4°C until stopped.

Annealing NLS.Serine (again)

3μL NLS.Serine.Fwd
3μL NLS.Serine.Rev
2μL 10x annealing buffer
12μL DH₂O

Put in boiling water in beaker (see above), allowed to boil for 2 mins, then removed beaker from heat block, allowing the water to slowly cool with the annealing reaction tube still in it.

07-21-2010 [top]

Nanodroping 5xGal promoter

Nanodrop of ligated annealed oligos: 5200.8 ng/μL, 1.32 260/280

Gel Extraction of 5xGal promoter

poured 2% agarose Gel for gel extraction, ran 2μL of the ligated annealing reaction in 13μL EB buffer, with two KB plus 1 ladders, one on each side.

Gel extraction performed, into an eppendorf weighing .9563g

[click to enlarge]

Nanodrop for cleaned-up gel purified 5xGalpt negative - ran a 2% E-gel to check. There appears to be something at the .16kb size.

[click to enlarge]

07-22-2010 [top]

Sequencing Results

Barstar samples 1, 2, 5, and 6 were confirmed to be correct.

The LacI sequence was confirmed, however we were unable to confirm the presence of the NLS because of its position at the end of the gene where sequencing results are generally unreliable. (NNNNN) Lac1R and Lac3R were poor matches because of what GENEWIZ calls "Early Termination".

Barnase had some serious issues. We are re-growing colonies following ligation and intend to submit some new PCR product as well as the original ADDGENE plasmid for sequencing in the near future.

The Gal4 DNA binding domain samples 1 and 2 were perfect except for a SNP around 320 (in the case of GAL DBD 1) for our sequence and 223 of the biobricks sequence.

Gal4 DBD sample 3 was unsuccessful.

Arabidopsis PCR

[click to enlarge]

FINALLY! EXP2 PROMOTER AMPLIFIED!

~1000kb

[click to enlarge]

Fragments were gel isolated and purified using the QIAgen Gel Purification Kit with the Gel Extraction protocol using a microcentrifuge.

07-23-2010 [top]

No colonies from last night's ligations.

7-24-10 (Saturday)

Successful ACC synthase transformation:

[click to enlarge]

PFX polymerase PCR

1μL 10mM MgSO4
2μL 10X pfx amplification buffer
2μL 10X PCRx enhancer solution
1μL F primer
1μL R primer
0.5μL pfx polymerase
2μL DNTP

9.5μL total

DNA: 21ng/μL

50 to 200μL DNA per reaction
5 reactions
4μL per reaction = 20μL sample total (MM)

9.5μL + 4μL = 13.5μL therefore 6.5μL H20 per reaction

MM

5μL 10nM MgSO4
10μL 10X pfx amplification buffer
10μL 10X PCRx enhancer solution
5μL F primer
5μL R primer
2.5μL pfx polymerase
10μL DNTP
20μL genomic DNA
32.5μL H20

total volume:100μL

Arabidopsis PCR

[click to enlarge]

Transformations

Thawed 2 turbo cells on ice to thaw
Resuspended EcR, RXRHm, RXRLc tubes from Mr.Gene into 50μL DH₂O (5μg into 50μL)
Pippetted .5μL plasmid from each of the 5 synthesis products (EcR, RXRHm, RXRLc, ACC, and ACT2LacOpt) into a labled eppendorf containing 20μL of turbo cells each
Pippetted 2μL of the relavent ligation reaction from Friday (Strep.Barnase 2 ligation, Strep.Barnase 1, Gal4 DBD lig) into eppendorfs containing 20μL turbo cells (barn1strep, barn2strep and Gal4DBD)
Chilled on ice for 15 mins
Put in 42°C heat bath for 30 secs
Put on ice for 2 mins
Added 200μL SOC medium (to equal 10x the amount of cells)
Set to shake at 37°C for 30 mins (longer for Act2pt because kan selective cultures need more time to become established before facing the harsher Kan selection medium
Streaked 20μL on LB+amp plates
Put in incubator to shake overnight
After 45 mins to 1.5 hours, streaked Act2LacOpt on Kan+LB plate, put in incubator to shake overnight.

Making LB+Kan Plates

made 4 LB+Kanamycin plates

  15μL Kanamycin
  30μL DH₂O to ease streaking

Streaked this on LB agar plates with glass beads, and then turned the plate upside down and let dry until it was ready for bacterial sequencing, and then those same beads were reused.

Transformation of pLAS and Terminator Resuspended the following from the registry with 10μL DH₂O

  p(LAS)TetO, on pSB2K3 (kan res.), from plate 2, sell 11N
  Terminator, on pSB1AK3 (kan and amp res.), from plate 1, well 4H

07-27-2010 [top]

Innoculations

RXRHuman, RXRLocust, Barn1strep, Barn2strep, ACC, and EcR

(these are all amp resistant)

6 colonies of each are inocculated, except for Barn1 and Barn2, of which 4 are innocculated each.
5mL LB+amp and a smear of each colony from a relevant plate

The same is done in LB+Kan for pLAS and Act2lacO promoter, which are Kanamycin resistant.

Redoing Barnase-strep Tag Ligation poured a 2% agarose gel to gel-purify the barnase from Barnase1 Xba Pst1 cleanup and Barnase2 Xba Pst1 cleanup, both from (7/23)

Gel ran empty except for DB ladders

Phosphatase Treating B15 Spe1/Pst1 Cleanup

to prevent religation of the whole vector

35μL B15 Spe1 Pst1 cleanup from 7/23
4μL 10x green FD buffer
1μL Alkaline phosphatase

07-28-2010 [top]

Minipreps

Miniprepped and Nanodroped the following:

Plasmid	Quantity (ng/μL)	260/280
Act2LacO 1	46.1	1.92
RXRHm 4	23.8	2.15
RXRHm 2	45.2	1.94
Terminator 6	172.8	1.93
Terminator 3	456.5	1.91
RXRLc 2	42.7	1.76
RXRLc 4	58.5	2.03
RXRLc 5	45.1	2.06
ACC 3	91.5	2.01
Barn 2 Strep 3	41.0	2.12
Barn 2 Strep 2	90.3	1.95
Barn 1 Strep 2	67.1	1.99
Barn 1 Strep 3	92.9	1.99
EcR 5	115.3	1.95
EcR 2	108.0	1.97
EcR 4	79.4	1.97
EcR 3	111.6	1.96
EcR 6	95.8	1.96

Barnase Digest Gel Extraction Again blank on the first attempt, tried again:

Success!

	2	5	8	9	10	18
DNA	KB+ ladder	15μL Barnase2 digest 1/5 dilution 7/23	20μL 1/3 dilution Barn2 digest cleanup 7/23	5μL 1/3 dilution Barn2 digest cleanup 7/23	20μL 1/3 dilution Barn2 digest cleanup 7/23	KB+ ladder

[click to enlarge]

07-29-2010 [top]

Digest of Wednesday's minipreps

Gel Extraction of Barnase insert

followed gel extraction protocol in QIAquick Spin Handbook

Nanodrops:

Plasmid	Quantity (ng/μL)	260/280
Tube #1	9.7	1.79
Tube #2	17.4	1.57
Tube #3	2.7	1.24

Samples 1-9

[click to enlarge]

Lane 1: ladder
Lane 2: ladder
Lane 3: ACC synthase 1
Lane 4: ACC synthase 2
Lane 5: Acc synthase 3
Lane 6: Terminator 1
Lane 7: Terminator 2
Lane 8: Terminator 3
Lane 9: Act2LacI 1
Lane 10: Act2LacI 2
Lane 11: Act2LacI 3
Lane 12: ladder

insert sizes:

ACC synthase: 300 to 350
Terminator: unknown
Act2LacI: ~1200

Samples 10-18

[click to enlarge]

Lane 1: empty
Lane 2: ladder
Lane 3: Barn1-strep1
Lane 4: Barn1-strep2
Lane 5: Barn1-strep3
Lane 6: Barn2-strep1
Lane 7: Barn2-strep2
Lane 8: Barn2-strep3
Lane 9: EcR2
Lane 10: EcR3
Lane 11: EcR4
Lane 12: ladder

insert sizes

Barn1-strep: <400bp
Barn2-strep: <400bp
Ecr: ~1000bp

Samples 19-24

[click to enlarge]

Lane 1: empty
Lane 2: ladder
Lane 3: empty
Lane 4: RxR-Hm 1
Lane 5: RxR-Hm 2
Lane 6: RxR-Hm 3
Lane 7: RxR-Lc 1
Lane 8: RxR-Lc 2
Lane 9: RxR-Lc 3
Lane 10: empty
Lane 11: empty
Lane 12: ladder

insert size

RxR-Lc: 600-700bp
RxR-Hm: 600-700bp

Summary

We see one appropriately sized band (~350bp) from the ACC synthase miniprep #1, the others did not have any bands. Therefore henceforth we will only use plasmid from miniprep #1 for our reactions.
Terminator size unknown at present.
For the Act2LacI promoter we saw in all three lanes the insert sized at ~1200bp. All three minipreps should be fine to use for future reactions.
The Barnase-strep lanes all displayed inserts of the correct size, ~350 to 400 bp. All minipreps should be good for future reactions.
The Ect. Receptor insert appears to be about the right size, >1000bp, although the ladder is a bit hard to decipher, all minipreps should be good for future reactions.
The RxR-Hm did not show any evidence of the presence of an insert.
The RxR-Lc did show evidence of the presence of an insert, although it may be a bit larger than the expected size (600-700bp). Blurriness of the ladder makes it difficult to decipher.

Nanodrop of Gal4DBD Minipreps (from the beginning of the summer

Plasmid	Quantity (ng/μL)	260/280
Gal4DBD #1	26.5	1.89
Gal4DBD #3	16.7	1.85
Gal4DBD #4	19.1	1.83
Gal4DBD #5	82.2	2.16

PCR of VP16 and Gal4DBD (preparing for yeast vector insertion) Gal4DBD

2μL DNTPs
.5μL phusion polymerase
4μL 5x phusion buffer
1μL 1/10 dilution of BB.Gal4DBD.Rev
1μL 1/10 dilution of Bam.Gal4.Fwd
1μL 1/2 dilution Gal4DBD #3 (made 1/2 dilution such that 1μL will be <10 ng/μL
μL 10.5μLDH₂O

4 tubes of each, 20μL each tube

VP16

10.5μL DH₂O
2μL DNTPs
.5μL phusion polymerase
4μL 5xphusion buffer
1μL 1/10 dilution of VP16.Hind.Fwd
1μL 1/10 dilution of VP16.Rev
1μL 1/10 dilution of VP16 #1

PCR program: VP16 Gal4

1= 98°C for 10:00

2= 98°C for :15

3= 50°C to 60°C for :30

4= 72°C for 1:30

5= goto 2, 29 times

6= 72°C for 10:10

7= 4°C forever

07-30-2010 [top]

PCR of VP16 and Gal4DBD

1	2	3	4	5	6	7	8	9	10	11	12
	KB+ Ladder	Gal4 DBD 1	Gal4 DBD 2	Gal4 DBD 3	Gal4 DBD 4	VP16 1	VP16 2	VP16 3	VP16 4	KB+ Ladder

[click to enlarge]

PCR again for VP16 Using pfx polymerase

1μL 1/10 dilution Hind.Fwd
1μL 1/10 dilution VP16.Rev
1μL DNTPs
6μL enhancer buffer
.5μL MgSO₄
.5μL PFX polymerase
1μL 1/10 dilution VP16 #1
5μL DH₂O
4μL amp(lification?) buffer

Running PCR on two reactions. There was not enough PFX for both to get .5μL, so only one of them is likely to actually amplify.

Second ACC + RxrHm digest gel

[click to enlarge]

yes to ACC (left) no RxrHm

08-02-2010 [top]

2% E-gel of VP16 PCR product from Friday

Lane
1	2	3	4	5	6	7	8	9	10	11	12
	KB+ Ladder		VP16 PCR product 1				VP16 PCR product 2			KB+ Ladder

[click to enlarge]

Gel extraction Gal4 spe bam 8/2 and EcR bam xba 8/2 gel extracted and purified.

Gal4 on the left, EcR on the right

[click to enlarge]

Nanodrop of Gal4 and EcR Gel Extractions

Plasmid	Quantity (ng/μL)	260/280
Gal4 (1)	1.4	-3.18
Gal4 (2)	7.2	4.59
EcR (1)	5.3	2.52
EcR (2)	5.6	2.12

VP16 PCR 2 60μL reaction:

1.5 μL pfx polymerase
3μL DNTPs
18μL enhancer buffer
1.5μL MgSO₄
3μL 1/10 dilution VP16.Hind.Fwd
3μL 1/10 dilution VP16.Rev
3μL 1/10 dilution of VP16 (1)
12μL amp buffer
5μL DH₂O

using program:

  1=94°C for 2:00
  2=94°C for :15
  3=68°C for :30
  4=Goto 2, 30 times
  5=4.0°C forever
  6=end

No PCR product was visible.

VP16 PCR.3 60μL reaction:

1.5 μL pfx polymerase
3μL DNTPs
18μL enhancer buffer
1.5μL MgSO₄
3μL 1/10 dilution VP16.Hind.Fwd
3μL 1/10 dilution VP16.Rev
3μL 1/10 dilution of VP16 (1)
12μL amp buffer
5μL DH₂O

Using modified program:

  1=94°C for 2:00
  2=94°C for :15
  3=60°C for :30
  4=70°C for :45
  5=Goto 2, 30 times
  6=4.0°C forever
  7=end

(see tomorrow's entry for gel image)

Ligations

  Gal4 into EcR
  Barnase into B15
  LTP into B11 (positive control from team allergy)

Gal4

3μL Gal4 spe bam gel pur. (2) 8/2
9μL EcR xba bam gel pur. (2) 8/2
2μL 10x T4 ligase buffer
5μL DH₂O
1μL T4 ligase

EcR control

9μL EcR xba bam gel pur. (2) 8/2
2μL 10x T4 ligase buffer
8μL DH₂O
1μL T4 ligase

Barnase

1.5μL Barnase digest gel pur. (1) 7/29
3μL B15 spe pst phosphorylated clean up
2μL 10x T4 ligase buffer
12.5μL DH₂O
1μL T4 ligase

B15 control

3μL B15 spe pst phosphorylated clean up
2μL 10x T4 ligase buffer
14μL DH₂O
1μL T4 ligase

LTP

2μL LTP
1μL B11 backbone
2μL 10x T4 ligase buffer
16μL DH₂O (should have been 14μL, the 16 was accidental)
1μL T4 ligase

B11 control

1μL B11 backbone
2μL 10x T4 ligase buffer
16μL DH₂O (should have been 14μL, the 16 was accidental)
1μL T4 ligase

Allowed tubes to sit at room temp for an hour, proceeded to transformation

08-03-2010 [top]

VP16 PCR from last night

[click to enlarge]

Yesterday's transformations

[click to enlarge]

Glycerol Stocks from 7-27-10

keeping Act2lac0pt 1, ACC synthase 1, EcR 2, RXRLc 2

added .5mL 80% glycerol to .5mL overnight cell culture of selected transformation, vortexed thoroughly, and placed in the -80°C freezer in team fence box.

Annealing 35s min promoter

3μL 100μM 35sminpromt.Rev
3μL 100μM 35sminpromt.Fwd
3μL .5M NaCl
3μL 10x PNK buffer
18μL DH₂O

placed eppendorf in boiling water for 2 mins, then removed water from heat and allowed water and eppendorf to slowly cool together.

08-04-2010 [top]

PCR of VP16.3 60μL reaction:

1.5 μL pfx polymerase
3μL DNTPs
18μL enhancer buffer
1.5μL MgSO₄
3μL 1/10 dilution VP16.Hind.Fwd
3μL 1/10 dilution VP16.Rev
3μL 1/10 dilution of VP16 (1)
12μL amp buffer
5μL DH₂O

Using program:

  1=94°C for 2:00
  2=94°C for :15
  3=60/68°C for :30
  4=68°C for :10
  5=Goto 2, 30 times
  6=4.0°C forever
  7=end

Digestion of Gal-EcR BamH1 and Nco1 12 digestions, one for each Gal-EcR miniprep Each:

1μL BamH1
1μL Nco1
1μL 10x FD green buffer
2-7μL EcR-Gal miniprep
5-0μL DH₂O, to bring total volume to 10μL

Ran a 1% gel of the digest

1	2	3	4	5	6	7	8	9	10	11	12	13	14
	EcR Digest 1	EcR Digest 2	EcR Digest 3	EcR Digest 5	EcR Digest 6	EcR Digest 7	EcR Digest 8	EcR Digest 9	EcR Digest 10	EcR Digest 11	EcR Digest 12		KB+ ladder

[

[click to enlarge]

Ligated EcR to Gal4 and Barnase to Strep

08-05-2010 [top]

Colonies from yesterday's ligations

[click to enlarge]

Gel extraction of EcR-Gal Bam Nco

applied 3x gel volume of QG buffer (in μL) to each respective tube
placed in 42°C bath until the gel was melted
added 1 gel volume of isopropanol to each, and vortexed
applied contents of each tube to a QIAquick spin collumn
spun for 1 min
added 500μL QG buffer (to dissolve any remaining agarose)
spun for 1 min
added 750μL PE buffer
spun for 1 min, discared flow-through
spun for another minute
placed QIAquick collumn in a fresh, labeled eppendorf tube, applied 50μL EB buffer to elute, and spun for 1 min

Nanodrop of Gal4 and EcR Gel Extractions

Plasmid	Quantity (ng/μL)	260/280
Gal-EcR (1)	3.1	2.45
Gal-EcR (2)	4.5	1.72
Gal-EcR (3)	3.0	11.56
Gal-EcR (4)	3.6	2.21

Digestions VP16

1μL xba
1μL Pst
5μL VP16(2) miniprep
1μL 10x FD green buffer
2μL DH₂O

5xGalUAS

1μL xba
1μL Pst
2μL 5xGal4UAS (from Karmella)
1μL 10x FD green buffer
5μL DH₂O

PhyB

1μL Bam
1μL Nco
16μL PhyB
2μL 10x FD green buffer

Sending EcR-Gal for sequencing

5μL of each primer, either fwd or rev, at 1/20 dilution
10μL of sample and DH₂O, such that sample is at 500ng total mass

Sealed tubes with parafilm. Submited Gal-EcR 1, 7, 10, and 12 (the one's with inserts on the digestion gel from 8/4)

08-06-2010 [top]

Digestions pACT2

1μL Bam
1μL Xho
6μL pACT2 (tube with a green top)
1μL FD Green buffer
1μL DH₂O

RXRLc

1μL Bam
1μL Xho
9μL RXRLc4
2μL FD Green buffer
7μL DH₂O

Barstar

1μL Eco
1μL Pst
2μL Barstar 3
1μL FD Green buffer
5μL DH₂O

Barnase

1μL Eco
1μL Pst
1.5μL Barnase 2
1μL FD Green buffer
5.5μL DH₂O

5xGalUAS

1μL Spe
1μL Pst
2μL 5xGalUAS (from Karmella)
1μL FD Green buffer
5μL DH₂O

pENT Cup

1μL Spe
1μL Pst
1μL pENT Cup
1μL FD Green buffer
6μL DH₂O

EcR-Gal

1μL Xba
1μL Pst
2μL EcR-Gal 7
1μL FD Green buffer
5μL DH₂O

VP16

1μL Spe
1μL Pst
5μL VP16(1)
1μL FD Green buffer
2μL DH₂O

RXRLc

1μL Xba
1μL Pst
9μL RXRLc4
2μL FD Green buffer
7μL DH₂O

V24

1μL Eco
1μL Pst
3μL V24
1μL FD Green buffer
4μL DH₂O

V26

1μL Eco
1μL Pst
9μL V26
12μL FD Green buffer
7μL DH₂O

PhyB

1μL Bam
1μL Nco
16μL PhyB
2μL FD Green buffer

08-09-2010 [top]

[click to enlarge]

yes colonies for ligation of exp2 and transformation of camv

Digests

[click to enlarge]

ladder
1. pACT2 (backbone)
2. RxRLC (insert)
3. Barstar (insert)
4. Barnase (insert)
5. 5XGalUAS (backbone)
6. PENTcup (backbone)
7. V26 (backbone)
8. Ecr-Galv (insert)

- - - no insert for 2 (RxRLC) or 8 (Ecr-Galv)

[click to enlarge]

ladder
RxRLC (Xba and Pst) (insert)
RxRLC (Bam and Xho) (insert) NO INSERT HERE
PhyB (backbone)
V24 (backbone)
VP16 (backbone)

08-10-2010 [top]

[Image: 810digestslater.jpg]]

ladder
PhyB (backbone)
Barstar 4 (insert)
V26 (backbone)
GalDBD1 (insert)
GalDBD5 (insert)
B21 (backbone)

Digests 8/10

PhyB with Bam and Nco
Barstar with Pst and Eco
V26 with Pst and Eco
Gal4DBD with Eco and Spe (Gal4DBD minipreps 1 and 5)
Arp2BB PCR product with Xba and Pst
Arp PCR product with Xba and Pst
B21 with Xba and Pst
ACC synthase with Eco and Spe
Barnase ligation product with Eco and Xba

08-11-2010 [top]

colonies from yesterday's transformations/ligations

[click to enlarge]

08-12-2010 [top]

[click to enlarge]

This picture is not the best picture but we did find colonies on the plate with the arp2/b21 transformation.

[click to enlarge]

transformation from karmella's stock

08-13-2010 [top]

Transformations from Yesterday's Ligations

[click to enlarge]

Innoculated colonies from EcR-Gal, Nost-pENT, and VP16Gal4DBD. No colonies visible on Nost-Act2LacO.

All Controls were clean or had very very few colonies (<2).

@@ Line 1: / Line 1: @@
 {{HarvardFancybox}}
 {{harvard fence}}
+{{harvard_notebook}}
 __NOTOC__
-==notebook==
+<html><h1><a name="notebook">notebook</a></h1></html>
 {| style="font-size:14px;text-align:center;width:550px;" border="0" cellspacing="6"
 |-
 | Week 1  || [[#06-14-2010|6-14-2010]] || [[#06-15-2010|06-15-2010]] || [[#06-16-2010|06-16-2010]] || [[#06-17-2010|06-17-2010]] || [[#06-18-2010|06-18-2010]]
 |-
-| Week 2  || [[#06-21-2010|06-21-2010]] || [[#06-22-2010|06-22-2010]] || [[#06-23-2010|06-23-2010]] || [[#06-24-2010|06-24-2010]] || [[#06-25-2010|06-25-2010]]
+| Week 2  || [[#06-21-2010|06-21-2010]] || [[#06-22-2010|06-22-2010]] || [[#06-23-2010|06-23-2010]] || [[#06-24-2010|06-24-2010]] || -
 |-
 | Week 3  || [[#06-28-2010|06-28-2010]] || [[#06-29-2010|06-29-2010]] || [[#06-30-2010|06-30-2010]] || [[#07-01-2010|07-01-2010]] || [[#07-02-2010|07-02-2010]]
 |-
-| Week 4  || [[#07-05-2010|07-05-2010]] || [[#07-06-2010|07-06-2010]] || [[#07-07-2010|07-07-2010]] || [[#07-08-2010|07-08-2010]] || [[#07-09-2010|07-09-2010]]
+| Week 4  || - || [[#07-06-2010|07-06-2010]] || [[#07-07-2010|07-07-2010]] || [[#07-08-2010|07-08-2010]] || [[#07-09-2010|07-09-2010]]
 |-
 | Week 5  || [[#07-12-2010|07-12-2010]] || [[#07-13-2010|07-13-2010]] || [[#07-14-2010|07-14-2010]] || [[#07-15-2010|07-15-2010]] || [[#07-16-2010|07-16-2010]]
 |-
-| Week 6  || [[#07-19-2010|07-19-2010]] || [[#07-20-2010|07-20-2010]] || [[#07-21-2010|07-21-2010]] || [[#07-22-2010|07-22-2010]] || [[#07-23-2010|07-23-2010]]
+| Week 6  || - || - || [[#07-21-2010|07-21-2010]] || [[#07-22-2010|07-22-2010]] || [[#07-23-2010|07-23-2010]]
 |-
-| Week 7  || [[#07-26-2010|07-26-2010]] || [[#07-27-2010|07-27-2010]] || [[#07-28-2010|07-28-2010]] || [[#07-29-2010|07-29-2010]] || [[#07-30-2010|07-30-2010]]
+| Week 7  || - || [[#07-27-2010|07-27-2010]] || [[#07-28-2010|07-28-2010]] || [[#07-29-2010|07-29-2010]] || [[#07-30-2010|07-30-2010]]
 |-
 | Week 8  || [[#08-02-2010|08-02-2010]] || [[#08-03-2010|08-03-2010]] || [[#08-04-2010|08-04-2010]] || [[#08-05-2010|08-05-2010]] || [[#08-06-2010|08-06-2010]]
@@ Line 26: / Line 27: @@
 |}
-==06-14-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="06-14-2010" class="date">06-14-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
 <b>LacI Transformation</b>
-Performed bacterial transformation according to [[Silver:_Bacterial_Transformation]], however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.
+Performed bacterial transformation according to [http://openwetware.org/wiki/Silver:_Bacterial_Transformation Silver Lab Bacterial Transformation], however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.
 Transformed the following, each into its own tube of TOP10 E.Coli:
@@ Line 43: / Line 44: @@
 Plates incubated overnight, colonies observed in all plates except the control.
+<html>
+<table>
+<tr>
+<td>
+<div>
+<a href="http://openwetware.org/images/6/65/Colonies_6-15-10.jpg" id="single_image"><img src="http://openwetware.org/images/6/65/Colonies_6-15-10.jpg" width="100px"/> [click to enlarge]</a>
+</div>
+</td>
-[[Image:Colonies 6-15-10.jpg|300px]]
+<td>
-[[Image:Colonies-2 6-15-10.jpg|300px]]
+<div>
-[[Image:Colonies-3 6-15-10.jpg|300px]]
+<a href="http://openwetware.org/images/1/1c/Colonies-2_6-15-10.jpg" id="single_image"><img src="http://openwetware.org/images/1/1c/Colonies-2_6-15-10.jpg" width="100px"/> [click to enlarge]</a>
+</div>
+</td>
-==06-15-2010 [ [[#top|top]] ]==
+<td>
+<div>
+<a href="http://openwetware.org/images/3/33/Colonies-3_6-15-10.jpg" id="single_image"><img src="http://openwetware.org/images/3/33/Colonies-3_6-15-10.jpg" width="100px"/> [click to enlarge]</a>
+</div>
+</td></tr></table>
+</html>
+<html><div id="entry"><span id="date"><a name="06-15-2010" class="date">06-15-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-===Oligonucleotides for LacI===
+'''Oligonucleotides for LacI'''<br>
 the following sequences for LacI and a Nuclear Localization Signal (NLS) were ordered for PCR:
@@ Line 67: / Line 85: @@
 '-CTA GAT CTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA -3'
-===VP16 transcription activating domain bacterial transformation===
+'''VP16 transcription activating domain bacterial transformation'''
 Performed bacterial transformation according to [[Silver:_Bacterial_Transformation]], however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.
@@ Line 82: / Line 100: @@
-===LacI Miniprep Preparations===
+'''LacI Miniprep Preparations'''<br>
 Prepared 6 overnight cell cultures, consisting of three colonies each from the LacI wt and LacI+rapid degradation tail E.Coli cells transformed previously.
@@ Line 93: / Line 111: @@
-==06-16-2010 [ [[#top|top]] ]==
+{{HarvardFancybox}}
-===LacI Miniprep===
+<html><div id="entry"><span id="date"><a name="06-16-2010" class="date">06-16-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''LacI Miniprep'''
 Performed miniprep of overnight cell cultures of 3 colonies each of wildtype LacI transformed bacteria and LacI+rapid degradation tail bacteria.
 Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].
-===LacI Nanodrop===
+'''LacI Nanodrop'''
 Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.
@@ Line 125: / Line 147: @@
 |}
-===LacI Plasmid Digest===
+'''LacI Plasmid Digest'''<br>
 We digested 15μL of each of the 6 samples with 1μLH<sub>2</sub>O, 1μL EcoR1, and 1μL Pst1 (high efficiency restriction enzymes).
@@ Line 138: / Line 160: @@
 |}
-[[Image:LacI digest Gel 2010-06-16 16hr 34min.jpg|500px]]
+<html>
+<div>
+<a href="http://openwetware.org/images/e/e4/LacI_digest_Gel_2010-06-16_16hr_34min.jpg" id="single_image"><img src="http://openwetware.org/images/e/e4/LacI_digest_Gel_2010-06-16_16hr_34min.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-===VP16 Colony Inoculation===
+'''VP16 Colony Inoculation'''<br>
 Prepared 3 10cc tubes each containing .5μL LB+amp and cells from one of the three colonies of the VP16 cell culture of the previous night (colonies from the second-tube plate, the cells in which were heatshocked in the correct order).
 Tubes were set to shake at 37°C overnight.
-===Preparing Glycerol Stocks of LacI wt and LacI+LVA===
+'''Preparing Glycerol Stocks of LacI wt and LacI+LVA'''<br>
 Prepared 2 cryo tubes, with the following labeling:
@@ Line 164: / Line 190: @@
 Both Cryo tubes were placed in the box labeled 'Genetic Fence iGEM 2010' in the -80°C freezer.
+<html><div id="entry"><span id="date"><a name="06-17-2010" class="date">06-17-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''VP16 Miniprep'''
-==06-17-2010 [ [[#top|top]] ]==
-===VP16 Miniprep===
 Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:
 *VP16<br>
-====Protocol====
+'''Protocol'''
 *4ml from each overnight cell culture of VP16 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>
@@ Line 192: / Line 215: @@
 *flow through kept and purity tested<br>
-===Nanodrop Values for VP16===
+'''Nanodrop Values for VP16'''
 *VP16 #1
@@ Line 206: / Line 229: @@
 **260/280 = 1.92
-===Digestion===
+'''Digestion'''
 Quantities per double digestion reaction
@@ Line 233: / Line 256: @@
 *1 unit enzyme cuts 1 μg DNA in 30-60 minutes
-===Digestion Gel===
+'''Digestion Gel'''
 Ingredients
@@ Line 270: / Line 293: @@
-===Bacterial transformation of GAL4 DNA binding domain===
+'''Bacterial transformation of GAL4 DNA binding domain'''
 BBa_K105007, plate 3 well 9I, psB1A2
@@ Line 284: / Line 307: @@
 *streaked on LB + amp paltes, incubated at 37°C overnight
+<html><div id="entry"><span id="date"><a name="06-18-2010" class="date">06-18-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==06-18-2010 [ [[#top|top]] ]==
+'''LacI+NLS PCR'''
-===LacI+NLS PCR===
 LacI+NLS primers arrived in mail
@@ Line 311: / Line 333: @@
 used LACIN program in phusion PCR machine to perform PCR
-==06-21-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="06-21-2010" class="date">06-21-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-===Barnase and Barstar===
+'''Barnase and Barstar'''
 Barnase and Barstar Plasmids from ADDGENE arrived in mail
@@ Line 330: / Line 352: @@
-===Notes from lab meeting===
+'''Notes from lab meeting'''
 Karmella suggested we try adding an LVA tail to the Cre we use for the crelox fence system.
-===GAL4 DBD Innoculation===
+'''GAL4 DBD Innoculation'''
 Pipetted 5mL of LB+Amp each into capped cc tubes, and mixed bacterial scrapings from 5 separate colonies of transformed GAL4 DNA binding domain E.Coli. Set to shake at 37 degrees C overnight.
-==06-22-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="06-22-2010" class="date">06-22-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-===GAL4DBD Miniprep===
+'''GAL4DBD Miniprep'''
+<html>
+<div>
+<a href="http://openwetware.org/images/1/12/062210_vector2.jpg" id="single_image"><img src="http://openwetware.org/images/1/12/062210_vector2.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+HindIII + PstI digest of miniprepped V9-V12 (#5,6) to check whether insert is present.]
-[[Image:062210_vector2.jpg|thumb|right|HindIII + PstI digest of miniprepped V9-V12 (#5,6) to check whether insert is present.]]
 *pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute
@@ Line 357: / Line 387: @@
 *tubes labeled "GAL4" plus the specific colony number
-===PCR===
+'''PCR'''
 PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)
@@ Line 375: / Line 405: @@
 =4°C for ∞
-[[Image:622file1.jpg|thumb|left|Lane 1:ladder, Lane 3:O2, Lane 4:E10. PCR succesful]]
-[[Image:622file1.jpg|left|Lane 1:ladder, Lane 3:O2, Lane 4:E10. PCR succesful]]
-[[Image:cutout622.jpg|thumb|right]]
+<html>
+<div>
+<a href="http://openwetware.org/images/8/85/File.jpg" id="single_image"><img src="http://openwetware.org/images/8/85/File.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+PCR successful
 PCR for E10 LacI
@@ Line 385: / Line 418: @@
 *The above settings on the PCR machine were used
-[[Image:cutout622.jpg|thumb|right]]
+<html>
+<div>
+<a href="http://openwetware.org/images/c/ce/Cutout622.jpg" id="single_image"><img src="http://openwetware.org/images/c/ce/Cutout622.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+Gel cut-out
 To Make Agarose Gel
 *150 mL TAE buffer combined with 1.5 g agarose in beaker
@@ Line 399: / Line 438: @@
 see [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2 QIAquick spin handbook]
-===Innoculations===
+'''Innoculations'''
 Colonies from the following cultures were innoculated and set to shake at 37°C overnight:
@@ Line 410: / Line 449: @@
 *lox71
+<html><div id="entry"><span id="date"><a name="06-23-2010" class="date">06-23-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==06-23-2010 [ [[#top|top]] ]==
+'''Minipreps'''<br>
-===Minipreps===
 The following colonies were minipreped after passing the night in the shaking incubator (37°C)
 * PMT413
@@ Line 422: / Line 461: @@
 * lox71
-===Nanodrops for above minipreps===
+'''Nanodrops for above minipreps'''
 Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.
@@ Line 482: / Line 521: @@
 |}
-==06-24-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="06-24-2010" class="date">06-24-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-===Primers for Barstar, Barnase, and Gal4DBD arrive===
+'''Primers for Barstar, Barnase, and Gal4DBD arrive'''
 Primers:
@@ Line 505: / Line 545: @@
-===Digestion===
+'''Digestion'''
 Our first digestion was unsuccessful except for the confirmation of the PMT316 fragment; this can be attributed to an incorrect combination of restriction enzymes.  The digestion was performed again in the afternoon using EcoRI + SpeI for the biobricked plasmids (lox71, GFP mut, lox66, cre recombinase, and firefly luciferase) and EcoRI + HindIII for PMT413.
 Gel Image, first digestion:
-[[Image:failgel624.jpg||350px]]
-PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)
+<html>
+<div>
+<a href="http://openwetware.org/images/d/da/Failgel624.jpg" id="single_image"><img src="http://openwetware.org/images/d/da/Failgel624.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)
+<html>
+<div>
+<a href="http://openwetware.org/images/5/56/624secondattempt.jpg" id="single_image"><img src="http://openwetware.org/images/5/56/624secondattempt.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-[[Image:624secondattempt.jpg||350px]]
 The length of the shortest bands were confirmed to be roughly the same length of the desired excerpt with the exception of Lox66 and Lox71 where it was determined that the inserts being only 34 bp had run off the gel, therefore the single band seen was the remaining plasmid.
-===Yeast Growth Assay===
+'''Yeast Growth Assay'''
 To assay the effect of Methoxyfenozide on yeast, three flasks containing 25μL DMSO (control), 2.5μL of 100mM Methoxyfenozide stock in 25mL DMSO to form the 1μM condition, and 25μL of 100mM Methoxyfenozide stock in 25mL DMSO to from the 10μM condition.
 Each beaker also contained y190 yeast strains and YPD medium.
@@ Line 552: / Line 600: @@
 |}
+<html><div id="entry"><span id="date"><a name="06-28-2010" class="date">06-28-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==06-25-2010 [ [[#top|top]] ]==
+'''B21 Innoculation'''
-===B21 Innoculation===
 Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.
-===PCR of Gal4DBD and Barnase, attempt 2===
+'''PCR of Gal4DBD and Barnase, attempt 2'''
 Ran 3 tubes of each for a total of 6.
 x Mastermix:
@@ Line 574: / Line 621: @@
 Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program
-===Gel of Gal4 DBD and Barnase from second PCR attempt===
+'''Gel of Gal4 DBD and Barnase from second PCR attempt'''
 Ran 1.2% E-gel of the 6 PCR product tubes.
@@ Line 607: / Line 654: @@
 |}
+<html>
+<div>
+<a href="http://openwetware.org/images/7/71/Gal4_Barnase_PCR_2nd.jpg" id="single_image"><img src="http://openwetware.org/images/7/71/Gal4_Barnase_PCR_2nd.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-[[Image:Gal4_Barnase_PCR_2nd.jpg|350px]]
+'''QIAQuick Purification of PCR Product'''<br>
-===QIAQuick Purification of PCR Product===
 Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.
 *as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)
@@ Line 621: / Line 671: @@
 *labeled the eppendorf tube 'Barstar PCR purified'
+<html><div id="entry"><span id="date"><a name="06-29-2010" class="date">06-29-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==06-28-2010 [ [[#top|top]] ]==
 B21 plasmid maxiprep
@@ Line 633: / Line 679: @@
 followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition)
+<html><div id="entry"><span id="date"><a name="06-30-2010" class="date">06-30-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==06-29-2010 [ [[#top|top]] ]==
+'''pMT1002 Miniprep'''
-===pMT1002 Miniprep===
 *4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>
@@ Line 654: / Line 700: @@
 Also see pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook] for miniprep instructions.
-===pMT1002 Miniprep Nanodrop values===
+'''pMT1002 Miniprep Nanodrop values'''
 {| border="3" style="margin-left: 3em;"
 |-
@@ Line 669: / Line 715: @@
 |}
-===PCR of miniprepped Barnase===
+'''PCR of miniprepped Barnase'''
 μL per tube
@@ Line 684: / Line 730: @@
 samples were made for the Barnase
-===pMT1002 Barnase PCR===
-[[Image:Pmt1002barnasePCRattempt1.jpg|350px]]
-===B21 Plasmid Digest===
-put 10x green FD buffer on ice to thaw.
-Tube A:
-*1μL Xba1
-*1μL Pst1
-*2μL green 10x FD buffer
-*3.4μL colony A plasmid (the amount used to get .5μg of plasmid)
-*12.6μL H<sub>2</sub>O (to bring the total to 20μL)
-Tube B:
-*1μL Xba1
-*1μL Pst1
-*2μL green 10x FD buffer
-*1.7μL colony B plasmid (the amount used to get .5μg of plasmid)
-*14.3μL H<sub>2</sub>O (to bring the total to 20μL)
-Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut
-[[Image:B21_digest_6-30-10.jpg|350px]]
-Second Attempt
-Tube A:
-*1μL Xba1
-*1μL Pst1
+'''B21 Plasmid Digest'''
-*5μL green 10x FD buffer
-*12.5μL colony A plasmid (the amount used to get .5μg of plasmid)
-*30.5μL H<sub>2</sub>O (to bring the total to 50μL)
-Tube B:
-*1μL Xba1
-*1μL Pst1
-*5μL green 10x FD buffer
-*6.6μL colony B plasmid (the amount used to get .5μg of plasmid)
-*36.4μL H<sub>2</sub>O (to bring the total to 50μL)
-Ran on a 1% agarose gel, 100 Volts for 30-40 mins.
-Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A
-[[Image:B21plasmiddigest2ndattempt.jpg|350px]]
-Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.
-===Other===
-*Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.
-*Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.
-==06-30-2010 [ [[#top|top]] ]==
-===pMT1002 Miniprep===
-*4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>
-*remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time<br>
-*excess LP+amp decanted, the pellet resuspended in 250μL P1<br>
-*contents transferred from each 15ml conical to a new epindorf tube<br>
-*250 μL P2 added to each tube, mixed by inverting gently 4-6 times<br>
-*350 μL N3 added to each tube, mixed by inverting as before<br>
-*tubes centrifuged for 10 minutes at 13,000 rpm<br>
-*supernatants pipetted into new QIAprep sin columns<br>
-*columns centrifuged for 30-60 seconds, flow through discarded<br>
-*QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds<br>
-*columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds<br>
-*flow through was discarded and tubes centrifuged for an additional minute<br>
-*column was placed in a clean 1.5 ml microcentrifuge tube<br>
-*50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after<br>
-*flow through kept, eppendorfs labeled pMT1002 plus their colony number<br>
-Also see pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook] for miniprep instructions.
-===pMT1002 Miniprep Nanodrop values===
-{| border="3" style="margin-left: 3em;"
-|-
-! Plasmid !! Quantity (ng/μL) !! 260/280
-|-
-! pMT1002 #1
-| 68.2 || 1.84
-|-
-! pMT1002 #2
-| 138.7 || 1.84
-|-
-! pMT1002 #3
-| 127.2 || 1.88
-|}
-===PCR of miniprepped Barnase===
-μL per tube
-In each tube:
-*1μL Forward primer
-*1μL Reverse primer
-*0.5μL polymerase
-*4μL 5X buffer
-*2μL DNTPs
-*1μL DNA sample
-*10.5μL water
-samples were made for the Barnase
-===pMT1002 Barnase PCR===
-[[Image:Pmt1002barnasePCRattempt1.jpg|350px]]
-===B21 Plasmid Digest===
 put 10x green FD buffer on ice to thaw.
@@ Line 839: / Line 758: @@
 *14.3μL H<sub>2</sub>O (to bring the total to 20μL)
 Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut
-[[Image:B21_digest_6-30-10.jpg|350px]]
+<html>
+<div>
+<a href="http://openwetware.org/images/f/f8/Pmt1002barnasePCRattempt1.jpg" id="single_image"><img src="http://openwetware.org/images/f/f8/Pmt1002barnasePCRattempt1.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
 Second Attempt
@@ Line 877: / Line 800: @@
 Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A
-[[Image:B21plasmiddigest2ndattempt.jpg|350px]]
+<html>
+<div>
+<a href="http://openwetware.org/images/4/44/B21_digest_6-30-10.jpg" id="single_image"><img src="http://openwetware.org/images/4/44/B21_digest_6-30-10.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
 Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.
-===Other===
+'''Other'''
 *Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.
 *Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.
+<html><div id="entry"><span id="date"><a name="07-01-2010" class="date">07-01-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-01-2010 [ [[#top|top]] ]==
 B21 Backbone Gel Extraction
 *followed QIAquick gel extraction handbook except melting temperature was 42°C instead of 50°C
@@ Line 905: / Line 833: @@
 only one band was visible in the gel for each reaction.  Julia forgot to put the ladder in so it was not possible to precisely determine the single band size, but judging from the position of the loading dye it seemed that the plasmid had only been cut once.
-===Ligation===
+'''Ligation'''
 Ligation performed on Barstar, NLS, and LacIN
@@ Line 944: / Line 872: @@
 *incubate at 37°C overnight
-==07-02-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="07-02-2010" class="date">07-02-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-===pMT1002, pMT413 Digest===
+'''pMT1002, pMT413 Digest'''
 Digested with EcoR1 and HindIII
@@ Line 955: / Line 884: @@
 Lane 6 pMT413
-[[Image:PMT1002_413ecoRHindIII7-2-1.jpg|350px]]
+<html>
+<div>
+<a href="http://openwetware.org/images/e/eb/PMT1002_413ecoRHindIII7-2-1.jpg" id="single_image"><img src="http://openwetware.org/images/e/eb/PMT1002_413ecoRHindIII7-2-1.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-===Barnase PCR===
+'''Barnase PCR'''
 Standard PCR measurements were used except that a new 50X dNTP mix was used in place of the 5X buffer used in all previous PCR reactions.  The amount of dH20 was adjusted for the new quantity of dNTP mix accordingly.
-[[Image:71barnasepcrweird.jpg]]
+<html>
+<div>
-==07-05-2010 [ [[#top|top]] ]==
+<a href="http://openwetware.org/images/b/b3/71barnasepcrweird.jpg" id="single_image"><img src="http://openwetware.org/images/b/b3/71barnasepcrweird.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html><div id="entry"><span id="date"><a name="07-06-2010" class="date">07-06-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-06-2010 [ [[#top|top]] ]==
 Inoculated 7 Barstar and 3 NLS colonies from Thursday's ligation.
@@ Line 980: / Line 915: @@
 *31μL H2O
-[[Image:76B21 digest gel.pdf]]
+<html>
+<div>
+<a href="http://openwetware.org/images/a/a4/76B21_digest_gel.pdf" id="single_image"><img src="http://openwetware.org/images/a/a4/76B21_digest_gel.pdf" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
 Digestion successful, larger bands removed for gel extraction.
@@ Line 986: / Line 925: @@
 Gel extraction performed according to QIAgen gel extraction protocol.
+<html><div id="entry"><span id="date"><a name="07-07-2010" class="date">07-07-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-07-2010 [ [[#top|top]] ]==
+'''Miniprep of Barastar and NLS Ligation products'''
-===Miniprep of Barastar and NLS Ligation products===
 Performed miniprep of overnight cell cultures of 7 colonies of Barstar, 3 colonies of NLS.
@@ Line 994: / Line 933: @@
 Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].
-[[Image:77LacINpcr.pdf|350px]]
+<html>
+<div>
+<a href="http://openwetware.org/images/7/71/77LacINpcr.pdf" id="single_image"><img src="http://openwetware.org/images/7/71/77LacINpcr.pdf" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-===Digestion of Barstar and NLS Ligation products===
+'''Digestion of Barstar and NLS Ligation products'''
 reactions of Barstar and 3 reactions of NLS
@@ Line 1,016: / Line 959: @@
-[[Image:77barstarnlsdigest1.pdf|350px]]
+<html>
+<div>
+<a href="http://openwetware.org/images/6/6b/77barstarnlsdigest1.pdf" id="single_image"><img src="http://openwetware.org/images/6/6b/77barstarnlsdigest1.pdf" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-===PCR of LacIN===
+'''PCR of LacIN'''
 reactions, each with:
 *1μL Fwd LacIN Primer
@@ Line 1,048: / Line 995: @@
-===PCR of Barnase and Gal4DBD===
+'''PCR of Barnase and Gal4DBD'''
 reactions each of Barnase from pMT413, Barnase from pMT1002, and Gal4
@@ Line 1,079: / Line 1,026: @@
 *10.5μL DH<sub>2</sub>O
+<html><div id="entry"><span id="date"><a name="07-08-2010" class="date">07-08-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-08-2010 [ [[#top|top]] ]==
+<html>
+<div>
-[[Image:78PCRgelGalBarnaseLac.pdf]]
+<a href="http://openwetware.org/images/b/b3/78PCRgelGalBarnaseLac.pdf" id="single_image"><img src="http://openwetware.org/images/b/b3/78PCRgelGalBarnaseLac.pdf" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
 After receiving the new primers we performed PCR on barnase vectors PMT413 and PMT1002, LacIN, as well as the GAL4 DNA binding domain.  The ladder is in lane 2, the GAL4 PCR products are in lanes 5 through 9, the barnase PMT413 lanes 10 through 14, and the LacIN PCR product is in the last 3 lanes.  After PCR was confirmed successful, we decided to gel purify the LacIN because of the multiple band sizes seen in the gel.  Below is the gel where LacIN was gel extracted, the chunk missing from the gel is where LacIN was identified to be.
-[[Image:78gelextractskeleton.pdf]]
+<html>
+<div>
+<a href="http://openwetware.org/images/4/46/78gelextractskeleton.pdf" id="single_image"><img src="http://openwetware.org/images/4/46/78gelextractskeleton.pdf" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
 Gel extraction was performed according to the QIAgen gel extraction kit protocol using a microcentrifuge.
-===Barstar Sequencing===
+'''Barstar Sequencing'''
 Submited plasmids from each of the 7 Barstar colonies for sequencing.
+<html><div id="entry"><span id="date"><a name="07-09-2010" class="date">07-09-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-09-2010 [ [[#top|top]] ]==
+'''Digest of yesterday's ligations'''
-===Digest of yesterday's ligations===
 LacIN - 65μL
 μL Xba1
@@ Line 1,136: / Line 1,090: @@
 Eppendorfs labeled "LacIN digest cleanup 7/9," "Gal4 digest cleanup 7/9," and "Barnase digest cleanup 7/9"
+'''7-10-10''' (Saturday)
+'''Innoculating colonies from Friday's ligations'''
+*Barnase, Gal4, LacIN
+*4 cc tubes each, 5mL LB+amp, set to shake at 37°C overnight
-==07-12-2010 [ [[#top|top]] ]==
+'''7-11-10''' (Sunday)
+'''Miniprep of Barnase, Gal4 and LacIN ligations'''
-==07-13-2010 [ [[#top|top]] ]==
+* No cells found in Barnase colonies 1 and 2 --> innoculations 3 and 4 renamed Barnase 1 and 2, replacing the cell-free cultures.
+*pipetted 4 ml from each overnight cell culture (excepting the cell-free Barnase cultures) into a 15 ml conical *centrifuged at 4400 rpm for 6 minute
+*remaining overnight cell cultures were placed in fridge
+*decanted LB+amp, resuspended cells in 250 μL P1 buffer
+*contents transferred to eppendorfs
+*250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times
+*350 μL N3 buffer added to each, tubes inverted 4-6 times
+*centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns
+*centrifuged for 30-60 seconds, flow through discarded
+*0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute
+*QIAprep columns put into new eppendorfs
+*50 μL buffer EB added to columns, let stand for 1 minute
+*centrifuged for 1 minute at 13,000 rpm
+<html><div id="entry"><span id="date"><a name="07-12-2010" class="date">07-12-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Nanodrop of Barnase, Gal4 and LacIN Minipreps'''
+{| border="3" style="margin-left: 3em;"
+|-
+! Plasmid !! Quantity (ng/μL) !! 260/280
+|-
+! Barnase 1
+| 226.1 ||
+|-
+! Barnase 2
+| 336.3 || 1.94
+|-
+! Gal4 1
+| 298.0 || 1.88
+|-
+! Gal4 2
+| 363.3 || 1.92
+|-
+! Gal4 3
+| 301.9 || 1.89
+|-
+! LacIN 1
+| 376.7 || 1.89
+|-
+! LacIN 2
+| 237.8 || 1.90
+|-
+! LacIN 3
+| 339.4 || 1.92
+|-
+! LacIN 4
+| 368.9 || 1.92
+|}
+'''Digestion of Ligation product minipreps'''
+Xba1 and Pst1
+Mastermix (10x)
+* 10μL Xba1
+* 10μL Pst1
+* 20μL Buffer
+Barnase 1
+μL Barnase 1
+μL DH<sub>2</sub>O
+μL Mastermix
+Barnase 2
+μL Barnase 2
+μL DH<sub>2</sub>O
+μL Mastermix
+Gal 1
+μL Gal 1
+μL DH<sub>2</sub>O
+μL Mastermix
+Gal 2
+.5μL Gal 2
+.5μL DH<sub>2</sub>O
+μL Mastermix
+Gal 3
+μL Gal 3
+μL DH<sub>2</sub>O
+μL Mastermix
+LacIN 1
+.5μL LacIN 1
+.5μL DH<sub>2</sub>O
+μL Mastermix
+LacIN 2
+μL LacIN 2
+μL DH<sub>2</sub>O
+μL Mastermix
+LacIN 3
+.5μL LacIN 1
+.5μL DH<sub>2</sub>O
+μL Mastermix
+LacIN 4
+.5μL LacIN 1
+.5μL DH<sub>2</sub>O
+μL Mastermix
+Put tubes in 37°C heat bath for 30 mins, then put them on ice.
+Gel of Digestions:
+<html>
+<div>
+<a href="http://openwetware.org/images/f/f9/07122010ve3ctordigestmirbraz2.jpg
+" id="single_image"><img src="http://openwetware.org/images/f/f9/07122010ve3ctordigestmirbraz2.jpg
+" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Sequencing'''
+*submitted Barnase 1 and 2, Gal4 1,2, and 3, and LacIN 1,2,3, and 4 for sequencing
+<html><div id="entry"><span id="date"><a name="07-13-2010" class="date">07-13-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+<html>
+<div>
+<a href="http://openwetware.org/images/5/5a/713gelyeastbackbone.jpeg" id="single_image"><img src="http://openwetware.org/images/5/5a/713gelyeastbackbone.jpeg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/6/6c/713gelyeastcutout.jpeg" id="single_image"><img src="http://openwetware.org/images/6/6c/713gelyeastcutout.jpeg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+The yeast backbones containing the PIF3 and PHYB inserts was isolated and purified.  The latter image shows where the appropriately sized bands were excised.
+Nanodrop values of final product are:
+PHYB 11ng/μL
+PIF3 20ng/μL
+<html><div id="entry"><span id="date"><a name="07-14-2010" class="date">07-14-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Phenol Chloroform DNA Extraction'''
+*resuspend DNA in approximately 200μL molecular grade pure water
+*add 1 volume phenol chloroform
+**when pipetting the phenol chloroform be sure to pipette from closer to the middle level of the liquid as the top two centimeters do not contain what we want
+*centrifuge at top speed for 5 minutes
+*collect the aqueous top layer and transfer to a clean microcentrifuge tube
+**the remaining liquid is toxic and must be carefully disposed of in the "toxic waste" bottle in the fume hood
+*add 1 volume phenol chloroform
+*centrifuge at top speed for 5 minutes
+*collect the aqueous top layer and put into a fresh tube
+*add three volumes 100% ethanol and 1/10 volume sodium acetate
+*allow solution to incubate on ice for 15 minutes
+*centrifuge at top speed for 10 minutes
+*pipette off the aqueous solution leaving a pellet of DNA in the bottom of the tube
+*wash in 500μL 70% ethanol
+*mix tube by inverting
+*centrifuge again for 10 minutes at top speed
+*air dry pellet by leaving tube open on bench (not on ice) til dry
+*after pellet has dried resuspend DNA in desired volume of H2O or buffer EB
+**presence of DNA can be confirmed by running a small sample on a gel
+'''NLS Ligations'''
+Ligation of NLS.Serine
+*2.5μL Backbone
+*1.5μL of 1/100 dilution (1.5ng NLS.Serine)
+*6μL H<sub>2</sub>O
+*10μL 2x quick ligase buffer
+*1μL quick ligase
+B21 control
+*2.5μL B21 Backbone
+*7.5μL DH<sub>2</sub>O
+*10μL 2x Quick Ligase Buffer
+*1μL Quick Ligase
+'''Transformation'''
+*1μL NLS lig into TOP10 cells
+*Chilled on ice for 30 mins
+*Heat shocked at 42°C for 30 seconds
+*Put on ice for 2 mins
+*Added 170μL SOC medium
+*Set to shake  in incubator for 30 mins
+*Streaked on LB+amp plates, incubated overnight
+'''Yeast backbone gel purified nanodrops'''
+{| border="1"
+! Sample
+! Concentraion in ng/μL
+! 260/280
+|-
+! 31 (1)
+| 8.5 || 3.00
+|-
+! 31(2)
+| 10.2 ||2.07
+|-
+! 32(3)
+| 11.1 ||2.31
+|-
+! 52(4)
+| 28.5 ||1.83
+|}
+<html><div id="entry"><span id="date"><a name="07-15-2010" class="date">07-15-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+Our previous attempt resulted in DNA with the following nanodrop values:
+*12.1ng/μL 260/280: 0.9
+*39.1ng/μL 260/280: 1.11
+It was believed that the other genomic DNA provided by the Matthews Lab before had similar values with regards to purity/contamination (also approaching 1)  Both team allergy and fence performed PCR multiple times unsuccessfully with these DNA samples.  It was thought that the low purity may be inhibiting the PCR.
+Today we used the QIAgen DNeasy Plant Mini Kit.  Liquid nitrogen was poured over the arabidopsis leaves in a mortar while they were ground with the pestle.  This produced a fine green powder of arabidopsis leaves from which genomic DNA was extracted starting with step seven of the QIAgen protocol following liquid nitrogen crushing.  The only change made to the protocol was that 40 μL of RNase 1 was added per reaction instead of the recommended 4 μL because the concentration of our supply was ten-fold lower.
+Nanodrop values were as follows:
+*6.7 ng/μL 260/280: 1.42
+*5 ng/μL 260/280: 1.48
+<html><div id="entry"><span id="date"><a name="07-16-2010" class="date">07-16-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''PCR of Arabidopsis Genomic DNA, attempt 2'''
+PCR using the newly extracted DNA was unsuccessful.
+The degradation signal from the Austrians was successfully inoculated and colonies were found on the agar plates.  Individual colonies from the plates were inoculated into tubes of 5 ml LB+Amp and placed in the shaking incubator overnight. (37°C)
+'''Making Glyerol Stocks'''
+Combined .5 mL 80% glycerol with .5mL overnight cell culture of: Barnase 3 (number changed to Barnase 1 from miniprep onward), LacIN1, and Gal2.
+'''7-17-10''' (Saturday)
+'''5xGal4 Promoter Annealing Product Ligation'''
+*Made a 1 in 1000 dilution of the annealing reaction
+{| border="1"
+|+ ''Nanodrop''
+!  !! ng/μL !! 260/280
+|-
+! 5xGalpt 1/1000 dilution
+| align="center" | 17.6 || 2.25
+|-
+|}
+For a ligation reaction, we want:
+*50ng of backbone
+*A 1:3 ratio of backbone:insert
+* 3(ng of backbone / basepairs of backbone) = (ng of insert / basepairs of insert)
+xGalpt is 180 bases long, B21 backbone is 3.2kb.
+So we want 8.4375 ng of plasmid
+To reach 8.4ng of 5xGalpt, made a 1 in 2000 dilution of the 5xGalpt Annealing product
+Ligation Reaction:
+*1μL 1/2000 5xGalpt
+*2.5μL B21 Backbone
+*6.5μL DH<sub>2</sub>O
+*1μL Quick Ligase
+*10μL 2x Quick Ligase Buffer
+B21 Control Ligation:
+*2.5μL B21 Backbone
+*7.5μL DH<sub>2</sub>O
+*1μL Quick Ligase
+*10μL 2x Quick Ligase Buffer
+Incubated at room temp for 15 mins, put on ice and immediately transformed.
+'''Transformations of 5xGalpt, NLS.Serine anneal, and B21 Backbone control'''
+*Thawed 3 Turbo chemically competent cells on ice
+*Pippeted 1μL of the ligation or annealing reaction into its own tube of turbo cells
+*Chilled on ice for 30 mins
+*Heatshocked in 42°C water on heat block for 30 seconds
+*Chilled on ice for 2 mins
+*Added 170μL SOC medium to each tube, set to shake at 37°C for 15 mins
+*Streaked on LB + amp plates, and left in incubator overnight
+'''7-18-10''' (Sunday)
+'''Re-doing 5xGalpt and NLS.Serine Annealing Reactions'''
+Set ~400ml of water to boil in a beaker on a heat block.
+Phosphorylating 5xGalpt
+*3μL 100μM oligo1.5xgal4
+*3μL 100μM oligo2.5xgal4
+*3μL 100μM oligo3.5xgal4
+*3μL 100μM oligo4.5xgal4
+*3μL 10x PNK buffer
+*2μL nM ATP
+*2μL t4 PNK
+*11μL DH<sub>2</sub>O
+put in 37°C waterbath for 90 mins
+Then added 4μL 5M NaCl to halt the reaction
+To get rid of the NaCL so that I can ligate the overlapping oligos:
+*added 3 volues of 100% ethanol (90μL), spun at top speed for 20 mins
+*pippetted off and discarded the supernatant, added 100μL of 70% ethanol, broke up the pellet, and spun for 5 mins
+*pippeted off supernatant, allowed to air dry.
+*added 2μL T4 ligase buffer, 1μL T4 ligase, and 17μL DH<sub>2</sub>O, using this mix to resuspend the pellet
+*set in PCR machine at 16°C for 12 hours, then 4°C until stopped.
+'''Annealing NLS.Serine (again)'''
+*3μL NLS.Serine.Fwd
+*3μL NLS.Serine.Rev
+*2μL 10x annealing buffer
+*12μL DH<sub>2</sub>O
+Put in boiling water in beaker (see above), allowed to boil for 2 mins, then removed beaker from heat block, allowing the water to slowly cool with the annealing reaction tube still in it.
+<html><div id="entry"><span id="date"><a name="07-21-2010" class="date">07-21-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Nanodroping 5xGal promoter'''
+Nanodrop of ligated annealed oligos:
+.8 ng/μL,   1.32 260/280
+'''Gel Extraction of 5xGal promoter'''
+poured 2% agarose Gel for gel extraction, ran 2μL of the ligated annealing reaction in 13μL EB buffer, with two KB plus 1 ladders, one on each side.
+Gel extraction performed, into an eppendorf weighing .9563g
+<html>
+<div>
+<a href="http://openwetware.org/images/9/91/5xGalprmt_gel_extract_07-21.jpg" id="single_image"><img src="http://openwetware.org/images/9/91/5xGalprmt_gel_extract_07-21.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+Nanodrop for cleaned-up gel purified 5xGalpt negative - ran a 2% E-gel to check. There appears to be something at the .16kb size.
+<html>
+<div>
+<a href="http://openwetware.org/images/a/a1/5xGalptGelPur.Check.7-21.jpg" id="single_image"><img src="http://openwetware.org/images/a/a1/5xGalptGelPur.Check.7-21.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html><div id="entry"><span id="date"><a name="07-22-2010" class="date">07-22-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Sequencing Results'''
+Barstar samples 1, 2, 5, and 6 were confirmed to be correct.
+The LacI sequence was confirmed, however we were unable to confirm the presence of the NLS because of its position at the end of the gene where sequencing results are generally unreliable. (NNNNN)  Lac1R and Lac3R were poor matches because of what GENEWIZ calls "Early Termination".
+Barnase had some serious issues. We are re-growing colonies following ligation and intend to submit some new PCR product as well as the original ADDGENE plasmid for sequencing in the near future.
+The Gal4 DNA binding domain samples 1 and 2 were perfect except for a SNP around 320 (in the case of GAL DBD 1) for our sequence and 223 of the biobricks sequence.
+Gal4 DBD sample 3 was unsuccessful.
+'''Arabidopsis PCR'''
+<html>
+<div>
+<a href="http://openwetware.org/images/2/2e/722pcrexp2sucess.jpg" id="single_image"><img src="http://openwetware.org/images/2/2e/722pcrexp2sucess.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+FINALLY! EXP2 PROMOTER AMPLIFIED!
+~1000kb
+<html>
+<div>
+<a href="http://openwetware.org/images/3/37/722pcrexp2sucess2.jpg" id="single_image"><img src="http://openwetware.org/images/3/37/722pcrexp2sucess2.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+Fragments were gel isolated and purified using the QIAgen Gel Purification Kit with the Gel Extraction protocol using a microcentrifuge.
+<html><div id="entry"><span id="date"><a name="07-23-2010" class="date">07-23-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+No colonies from last night's ligations.
+'''7-24-10''' (Saturday)
+Successful ACC synthase transformation:
+<html>
+<div>
+<a href="http://openwetware.org/images/5/58/725acc.jpg" id="single_image"><img src="http://openwetware.org/images/5/58/725acc.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+PFX polymerase PCR
+*1μL 10mM MgSO4
+*2μL 10X pfx amplification buffer
+*2μL 10X PCRx enhancer solution
+*1μL F primer
+*1μL R primer
+*0.5μL pfx polymerase
+*2μL DNTP
+.5μL total
+DNA: 21ng/μL
+*50 to 200μL DNA per reaction
+*5 reactions
+*4μL per reaction = 20μL sample total (MM)
+.5μL + 4μL = 13.5μL
+therefore 6.5μL H20 per reaction
+MM
+*5μL 10nM MgSO4
+*10μL 10X pfx amplification buffer
+*10μL 10X PCRx enhancer solution
+*5μL F primer
+*5μL R primer
+*2.5μL pfx polymerase
+*10μL DNTP
+*20μL genomic DNA
+*32.5μL H20
+total volume:100μL
+'''Arabidopsis PCR'''
+<html>
+<div>
+<a href="http://openwetware.org/images/d/d5/726arapcregel.jpg-px240" id="single_image"><img src="http://openwetware.org/images/d/d5/726arapcregel.jpg-px240" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Transformations'''
+*Thawed 2 turbo cells on ice to thaw
+*Resuspended EcR, RXRHm, RXRLc tubes from Mr.Gene into 50μL DH<sub>2</sub>O (5μg into 50μL)
+*Pippetted .5μL plasmid from each of the 5 synthesis products (EcR, RXRHm, RXRLc, ACC, and  ACT2LacOpt) into a labled eppendorf containing 20μL of turbo cells each
+*Pippetted 2μL of the relavent ligation reaction from Friday (Strep.Barnase 2 ligation, Strep.Barnase 1, Gal4 DBD lig) into eppendorfs containing 20μL turbo cells (barn1strep, barn2strep and Gal4DBD)
+*Chilled on ice for 15 mins
+*Put in 42°C heat bath for 30 secs
+*Put on ice for 2 mins
+*Added 200μL SOC medium (to equal 10x the amount of cells)
+*Set to shake at 37°C for 30 mins (longer for Act2pt because kan selective cultures need more time to become established before facing the harsher Kan selection medium
+*Streaked 20μL on LB+amp plates
+*Put in incubator to shake overnight
+*After 45 mins to 1.5 hours, streaked Act2LacOpt on Kan+LB plate, put in incubator to shake overnight.
+'''Making LB+Kan Plates'''
+*made 4 LB+Kanamycin plates
+μL Kanamycin
+μL DH<sub>2</sub>O to ease streaking
+Streaked this on LB agar plates with glass beads, and then turned the plate upside down and let dry until it was ready for bacterial sequencing, and then those same beads were reused.
+'''Transformation of pLAS and Terminator'''
+Resuspended the following from the registry with 10μL DH<sub>2</sub>O
+   p(LAS)TetO, on pSB2K3 (kan res.), from plate 2, sell 11N
+   Terminator, on pSB1AK3 (kan and amp res.), from plate 1, well 4H
+<html><div id="entry"><span id="date"><a name="07-27-2010" class="date">07-27-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Innoculations'''
+*RXRHuman, RXRLocust, Barn1strep, Barn2strep, ACC, and EcR
+(these are all amp resistant)
+*6 colonies of each are inocculated, except for Barn1 and Barn2, of which 4 are innocculated each.
+*5mL LB+amp and a smear of each colony from a relevant plate
+The same is done in LB+Kan for pLAS and Act2lacO promoter, which are Kanamycin resistant.
+'''Redoing Barnase-strep Tag Ligation'''
+poured a 2% agarose gel to gel-purify the barnase from  Barnase1 Xba Pst1 cleanup and Barnase2 Xba Pst1 cleanup, both from (7/23)
+*Gel ran empty except for DB ladders
+'''Phosphatase Treating B15 Spe1/Pst1 Cleanup'''
+*to prevent religation of the whole vector
+*35μL B15 Spe1 Pst1 cleanup from 7/23
+*4μL 10x green FD buffer
+*1μL Alkaline phosphatase
+<html><div id="entry"><span id="date"><a name="07-28-2010" class="date">07-28-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Minipreps'''
+Miniprepped and Nanodroped the following:
+{| border="3" style="margin-left: 3em;"
+|-
+! Plasmid !! Quantity (ng/μL) !! 260/280
+|-
+! Act2LacO 1
+| 46.1 || 1.92
+|-
+! RXRHm 4
+| 23.8 || 2.15
+|-
+! RXRHm 2
+| 45.2 || 1.94
+|-
+! Terminator 6
+| 172.8 || 1.93
+|-
+! Terminator 3
+| 456.5 || 1.91
+|-
+! RXRLc 2
+| 42.7 || 1.76
+|-
+! RXRLc 4
+| 58.5 || 2.03
+|-
+! RXRLc 5
+| 45.1 || 2.06
+|-
+! ACC 3
+| 91.5 || 2.01
+|-
+! Barn 2 Strep 3
+| 41.0 || 2.12
+|-
+! Barn 2 Strep 2
+| 90.3 || 1.95
+|-
+! Barn 1 Strep 2
+| 67.1 || 1.99
+|-
+! Barn 1 Strep 3
+| 92.9 || 1.99
+|-
+! EcR 5
+| 115.3 || 1.95
+|-
+! EcR 2
+| 108.0 || 1.97
+|-
+! EcR 4
+| 79.4|| 1.97
+|-
+! EcR 3
+| 111.6 || 1.96
+|-
+! EcR 6
+| 95.8 || 1.96
+|-
+|}
+'''Barnase Digest Gel Extraction'''
+Again blank on the first attempt, tried again:
+Success!
+{| border="1"
+!  !! 2 !! 5 !! 8 !! 9 !! 10 !! 18
+|-
+! DNA
+| KB+ ladder || 15μL Barnase2 digest 1/5 dilution 7/23 || 20μL 1/3 dilution Barn2 digest cleanup 7/23 || 5μL 1/3 dilution Barn2 digest cleanup 7/23 || 20μL 1/3 dilution Barn2 digest cleanup 7/23 || KB+ ladder
+|-
+|}
+<html>
+<div>
+<a href="http://openwetware.org/images/f/fe/Barnase_pstxba_gel_ext_7-28.jpg" id="single_image"><img src="http://openwetware.org/images/f/fe/Barnase_pstxba_gel_ext_7-28.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html><div id="entry"><span id="date"><a name="07-29-2010" class="date">07-29-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Digest of Wednesday's minipreps'''
+'''Gel Extraction of Barnase insert'''
+*followed gel extraction protocol in QIAquick Spin Handbook
+Nanodrops:
+{| border="3" style="margin-left: 3em;"
+|-
+! Plasmid !! Quantity (ng/μL) !! 260/280
+|-
+! Tube #1
+| 9.7 || 1.79
+|-
+! Tube #2
+| 17.4 || 1.57
+|-
+! Tube #3
+| 2.7 || 1.24
+|-
+|}
+'''Samples 1-9'''
+<html>
+<div>
+<a href="hhttp://openwetware.org/images/b/b6/729_digest_1-9.jpg" id="single_image"><img src="http://openwetware.org/images/b/b6/729_digest_1-9.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+*Lane 1: ladder
+*Lane 2: ladder
+*Lane 3: ACC synthase 1
+*Lane 4: ACC synthase 2
+*Lane 5: Acc synthase 3
+*Lane 6: Terminator 1
+*Lane 7: Terminator 2
+*Lane 8: Terminator 3
+*Lane 9: Act2LacI 1
+*Lane 10: Act2LacI 2
+*Lane 11: Act2LacI 3
+*Lane 12: ladder
+insert sizes:
+*ACC synthase: 300 to 350
+*Terminator: unknown
+*Act2LacI: ~1200
+'''Samples 10-18'''
+<html>
+<div>
+<a href="http://openwetware.org/images/1/1a/729_digest_10-18.jpg" id="single_image"><img src="http://openwetware.org/images/1/1a/729_digest_10-18.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+*Lane 1: empty
+*Lane 2: ladder
+*Lane 3: Barn1-strep1
+*Lane 4: Barn1-strep2
+*Lane 5: Barn1-strep3
+*Lane 6: Barn2-strep1
+*Lane 7: Barn2-strep2
+*Lane 8: Barn2-strep3
+*Lane 9: EcR2
+*Lane 10: EcR3
+*Lane 11: EcR4
+*Lane 12: ladder
+insert sizes
+*Barn1-strep: <400bp
+*Barn2-strep: <400bp
+*Ecr: ~1000bp
+'''Samples 19-24'''
+<html>
+<div>
+<a href="http://openwetware.org/images/b/b8/729_digest_19-24.jpg" id="single_image"><img src="http://openwetware.org/images/b/b8/729_digest_19-24.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+*Lane 1: empty
+*Lane 2: ladder
+*Lane 3: empty
+*Lane 4: RxR-Hm 1
+*Lane 5: RxR-Hm 2
+*Lane 6: RxR-Hm 3
+*Lane 7: RxR-Lc 1
+*Lane 8: RxR-Lc 2
+*Lane 9: RxR-Lc 3
+*Lane 10: empty
+*Lane 11: empty
+*Lane 12: ladder
+insert size
+*RxR-Lc: 600-700bp
+*RxR-Hm: 600-700bp
+'''Summary'''
+*We see one appropriately sized band (~350bp) from the ACC synthase miniprep #1, the others did not have any bands.  Therefore henceforth we will only use plasmid from miniprep #1 for our reactions.
+*Terminator size unknown at present.
+*For the Act2LacI promoter we saw in all three lanes the insert sized at ~1200bp.  All three minipreps should be fine to use for future reactions.
+*The Barnase-strep lanes all displayed inserts of the correct size, ~350 to 400 bp.  All minipreps should be good for future reactions.
+*The Ect. Receptor insert appears to be about the right size, >1000bp, although the ladder is a bit hard to decipher, all minipreps should be good for future reactions.
+*The RxR-Hm did not show any evidence of the presence of an insert.
+*The RxR-Lc did show evidence of the presence of an insert, although it may be a bit larger than the expected size (600-700bp).  Blurriness of the ladder makes it difficult to decipher.
+'''Nanodrop of Gal4DBD Minipreps (from the beginning of the summer'''
+{| border="3" style="margin-left: 3em;"
+|-
+! Plasmid !! Quantity (ng/μL) !! 260/280
+|-
+! Gal4DBD #1
+| 26.5 || 1.89
+|-
+! Gal4DBD #3
+| 16.7 || 1.85
+|-
+! Gal4DBD #4
+| 19.1 || 1.83
+|-
+! Gal4DBD #5
+| 82.2 || 2.16
+|-
+|}
+'''PCR of VP16 and Gal4DBD (preparing for yeast vector insertion)'''
+Gal4DBD
+*2μL DNTPs
+*.5μL phusion polymerase
+*4μL 5x phusion buffer
+*1μL 1/10 dilution of BB.Gal4DBD.Rev
+*1μL 1/10 dilution of Bam.Gal4.Fwd
+*1μL 1/2 dilution Gal4DBD #3 (made 1/2 dilution such that 1μL will be <10 ng/μL
+*μL 10.5μLDH<sub>2</sub>O
+tubes of each, 20μL each tube
+VP16
+*10.5μL DH<sub>2</sub>O
+*2μL DNTPs
+*.5μL phusion polymerase
+*4μL 5xphusion buffer
+*1μL 1/10 dilution of VP16.Hind.Fwd
+*1μL 1/10 dilution of VP16.Rev
+*1μL 1/10 dilution of VP16 #1
+PCR program:
+VP16 Gal4
+= 98°C for 10:00
+= 98°C for :15
+= 50°C to 60°C for :30
+= 72°C for 1:30
+= goto 2, 29 times
+= 72°C for 10:10
+= 4°C forever
+<html><div id="entry"><span id="date"><a name="07-30-2010" class="date">07-30-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''PCR of VP16 and Gal4DBD'''
+{| class="wikitable" border ="1px solid black"
+|-
+!1
+!2
+!3
+!4
+!5
+!6
+!7
+!8
+!9
+!10
+!11
+!12
+|-
+|
+|KB+ Ladder
+|Gal4 DBD 1
+|Gal4 DBD 2
+|Gal4 DBD 3
+|Gal4 DBD 4
+|VP16 1
+|VP16 2
+|VP16 3
+|VP16 4
+|KB+ Ladder
+|
+|-
+|}
+<html>
+<div>
+<a href="http://openwetware.org/images/4/4c/PCR_VP16_Gal4DBD_7-30.jpg" id="single_image"><img src="http://openwetware.org/images/4/4c/PCR_VP16_Gal4DBD_7-30.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''PCR again for VP16'''
+Using pfx polymerase
+*1μL 1/10 dilution Hind.Fwd
+*1μL 1/10 dilution VP16.Rev
+*1μL DNTPs
+*6μL enhancer buffer
+*.5μL MgSO<sub>4</sub>
+*.5μL PFX polymerase
+*1μL 1/10 dilution VP16 #1
+*5μL DH<sub>2</sub>O
+*4μL amp(lification?) buffer
+Running PCR on two reactions. There was not enough PFX for both to get .5μL, so only one of them is likely to actually amplify.
+'''Second ACC + RxrHm digest gel'''
+<html>
+<div>
+<a href="http://openwetware.org/images/3/36/Sucesspcrkfkdigestacvtually.jpg" id="single_image"><img src="http://openwetware.org/images/3/36/Sucesspcrkfkdigestacvtually.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+yes to ACC (left) no RxrHm
+<html><div id="entry"><span id="date"><a name="08-02-2010" class="date">08-02-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''2% E-gel of VP16 PCR product from Friday'''
+{| class="wikitable" border ="1px solid black"
+|-
+!Lane
+|-
+|1
+|2
+|3
+|4
+|5
+|6
+|7
+|8
+|9
+|10
+|11
+|12
+|-
+|
+|KB+ Ladder
+|
+|VP16 PCR product 1
+|
+|
+|
+|VP16 PCR product 2
+|
+|
+|KB+ Ladder
+|
+|-
+|}
+<html>
+<div>
+<a href="http://openwetware.org/images/e/ef/VP16_PCR.2_8-1.jpg" id="single_image"><img src="http://openwetware.org/images/e/ef/VP16_PCR.2_8-1.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Gel extraction'''
+Gal4 spe bam 8/2 and EcR bam xba 8/2 gel extracted and purified.
+Gal4 on the left, EcR on the right
+<html>
+<div>
+<a href="http://openwetware.org/images/f/fc/Gal4_Ecr_digest_gel_ex_8-2.jpg" id="single_image"><img src="http://openwetware.org/images/f/fc/Gal4_Ecr_digest_gel_ex_8-2.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Nanodrop of Gal4 and EcR Gel Extractions'''
+{| border="3" style="margin-left: 3em;"
+|-
+! Plasmid !! Quantity (ng/μL) !! 260/280
+|-
+! Gal4 (1)
+| 1.4 || -3.18
+|-
+! Gal4 (2)
+| 7.2 || 4.59
+|-
+! EcR (1)
+| 5.3 || 2.52
+|-
+! EcR (2)
+| 5.6 || 2.12
+|}
+'''VP16 PCR 2'''
+μL reaction:
+*1.5 μL pfx polymerase
+*3μL DNTPs
+*18μL enhancer buffer
+*1.5μL MgSO<sub>4</sub>
+*3μL 1/10 dilution VP16.Hind.Fwd
+*3μL 1/10 dilution VP16.Rev
+*3μL 1/10 dilution of VP16 (1)
+*12μL amp buffer
+*5μL DH<sub>2</sub>O
+using program:
+=94°C for 2:00
+=94°C for :15
+=68°C for :30
+=Goto 2, 30 times
+=4.0°C forever
+=end
+No PCR product was visible.
+'''VP16 PCR.3'''
+μL reaction:
+*1.5 μL pfx polymerase
+*3μL DNTPs
+*18μL enhancer buffer
+*1.5μL MgSO<sub>4</sub>
+*3μL 1/10 dilution VP16.Hind.Fwd
+*3μL 1/10 dilution VP16.Rev
+*3μL 1/10 dilution of VP16 (1)
+*12μL amp buffer
+*5μL DH<sub>2</sub>O
+Using modified program:
+=94°C for 2:00
+=94°C for :15
+=60°C for :30
+=70°C for :45
+=Goto 2, 30 times
+=4.0°C forever
+=end
+(see tomorrow's entry for gel image)
+'''Ligations'''<br>
+   Gal4 into EcR
+   Barnase into B15
+   LTP into B11 (positive control from team allergy)
+Gal4
+*3μL Gal4 spe bam gel pur. (2) 8/2
+*9μL EcR xba bam gel pur. (2) 8/2
+*2μL 10x T4 ligase buffer
+*5μL DH<sub>2</sub>O
+*1μL T4 ligase
+EcR control
+*9μL EcR xba bam gel pur. (2) 8/2
+*2μL 10x T4 ligase buffer
+*8μL DH<sub>2</sub>O
+*1μL T4 ligase
+Barnase
+*1.5μL Barnase digest gel pur. (1) 7/29
+*3μL B15 spe pst phosphorylated clean up
+*2μL 10x T4 ligase buffer
+*12.5μL DH<sub>2</sub>O
+*1μL T4 ligase
+B15 control
+*3μL B15 spe pst phosphorylated clean up
+*2μL 10x T4 ligase buffer
+*14μL DH<sub>2</sub>O
+*1μL T4 ligase
+LTP
+*2μL LTP
+*1μL B11 backbone
+*2μL 10x T4 ligase buffer
+*16μL DH<sub>2</sub>O (should have been 14μL, the 16 was accidental)
+*1μL T4 ligase
+B11 control
+*1μL B11 backbone
+*2μL 10x T4 ligase buffer
+*16μL DH<sub>2</sub>O (should have been 14μL, the 16 was accidental)
+*1μL T4 ligase
+Allowed tubes to sit at room temp for an hour, proceeded to transformation
+<html><div id="entry"><span id="date"><a name="08-03-2010" class="date">08-03-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+VP16 PCR from last night
+<html>
+<div>
+<a href="http://openwetware.org/images/9/9f/83gelpcrvp16.jpg" id="single_image"><img src="http://openwetware.org/images/9/9f/83gelpcrvp16.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Yesterday's transformations'''
+<html>
+<div>
+<a href="http://openwetware.org/images/b/b4/Gal4-EcR_lig_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/b/b4/Gal4-EcR_lig_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/7/7b/EcR_neg_control_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/7/7b/EcR_neg_control_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/1/16/LTP_lig-1_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/1/16/LTP_lig-1_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="hhttp://openwetware.org/images/8/85/LTP_lig-2_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/8/85/LTP_lig-2_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/3/38/B11_neg_cont_lig_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/3/38/B11_neg_cont_lig_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/7/71/Barn-strep_lig_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/7/71/Barn-strep_lig_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/7/71/Barn-strep_lig_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/7/71/Barn-strep_lig_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/e/e7/B15_cont_lig_8-3.jpg" id="single_image"><img src="http://openwetware.org/images/e/e7/B15_cont_lig_8-3.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Glycerol Stocks'''
+from 7-27-10
+keeping Act2lac0pt 1, ACC synthase 1, EcR 2, RXRLc 2
+added .5mL 80% glycerol to .5mL overnight cell culture of selected transformation, vortexed thoroughly, and placed in the -80°C freezer in team fence box.
+'''Annealing 35s min promoter'''
+*3μL 100μM 35sminpromt.Rev
+*3μL 100μM 35sminpromt.Fwd
+*3μL .5M NaCl
+*3μL 10x PNK buffer
+*18μL DH<sub>2</sub>O
+placed eppendorf in boiling water for 2 mins, then removed water from heat and allowed water and eppendorf to slowly cool together.
+<html><div id="entry"><span id="date"><a name="08-04-2010" class="date">08-04-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''PCR of VP16.3'''
+μL reaction:
+*1.5 μL pfx polymerase
+*3μL DNTPs
+*18μL enhancer buffer
+*1.5μL MgSO<sub>4</sub>
+*3μL 1/10 dilution VP16.Hind.Fwd
+*3μL 1/10 dilution VP16.Rev
+*3μL 1/10 dilution of VP16 (1)
+*12μL amp buffer
+*5μL DH<sub>2</sub>O
+Using program:
+=94°C for 2:00
+=94°C for :15
+=60/68°C for :30
+=68°C for :10
+=Goto 2, 30 times
+=4.0°C forever
+=end
+'''Digestion of Gal-EcR'''
+BamH1 and Nco1
+digestions, one for each Gal-EcR miniprep
+Each:
+*1μL BamH1
+*1μL Nco1
+*1μL 10x FD green buffer
+*2-7μL EcR-Gal miniprep
+*5-0μL DH<sub>2</sub>O, to bring total volume to 10μL
+Ran a 1% gel of the digest
+{| class="wikitable" border ="1px solid black"
+|-
+|1
+|2
+|3
+|4
+|5
+|6
+|7
+|8
+|9
+|10
+|11
+|12
+|13
+|14
+|-
+|
+|EcR Digest 1
+|EcR Digest 2
+|EcR Digest 3
+|EcR Digest 5
+|EcR Digest 6
+|EcR Digest 7
+|EcR Digest 8
+|EcR Digest 9
+|EcR Digest 10
+|EcR Digest 11
+|EcR Digest 12
+|
+|KB+ ladder
+|-
+|}
+[<html>
+<div>
+<a href="http://openwetware.org/images/a/a3/EcR-Gal_lig_VP16.4_Pcr_8-04.jpg" id="single_image"><img src="http://openwetware.org/images/a/a3/EcR-Gal_lig_VP16.4_Pcr_8-04.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Ligated EcR to Gal4 and Barnase to Strep'''
+ <html><div id="entry"><span id="date"><a name="08-05-2010" class="date">08-05-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Colonies from yesterday's ligations'''
+<html>
+<div>
+<a href="http://openwetware.org/images/0/0c/84_colonies_1.jpg" id="single_image"><img src="http://openwetware.org/images/0/0c/84_colonies_1.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/1/1b/84_colonies_2.jpg" id="single_image"><img src="http://openwetware.org/images/1/1b/84_colonies_2.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+'''Gel extraction of EcR-Gal Bam Nco'''
+*applied 3x gel volume of QG buffer (in μL) to each respective tube
+*placed in 42°C bath until the gel was melted
+*added 1 gel volume of isopropanol to each, and vortexed
+*applied contents of each tube to a QIAquick spin collumn
+*spun for 1 min
+*added 500μL QG buffer (to dissolve any remaining agarose)
+*spun for 1 min
+*added 750μL PE buffer
+*spun for 1 min, discared flow-through
+*spun for another minute
+*placed QIAquick collumn in a fresh, labeled eppendorf tube, applied 50μL EB buffer to elute, and spun for 1 min
+'''Nanodrop of Gal4 and EcR Gel Extractions'''
+{| border="3" style="margin-left: 3em;"
+|-
+! Plasmid !! Quantity (ng/μL) !! 260/280
+|-
+! Gal-EcR (1)
+| 3.1 || 2.45
+|-
+! Gal-EcR (2)
+| 4.5 || 1.72
+|-
+! Gal-EcR (3)
+| 3.0 || 11.56
+|-
+! Gal-EcR (4)
+| 3.6 || 2.21
+|}
+'''Digestions'''
+VP16
+*1μL xba
+*1μL Pst
+*5μL VP16(2) miniprep
+*1μL 10x FD green buffer
+*2μL DH<sub>2</sub>O
+xGalUAS
+*1μL xba
+*1μL Pst
+*2μL 5xGal4UAS (from Karmella)
+*1μL 10x FD green buffer
+*5μL DH<sub>2</sub>O
+PhyB
+*1μL Bam
+*1μL Nco
+*16μL PhyB
+*2μL 10x FD green buffer
+'''Sending EcR-Gal for sequencing'''
+*5μL of each primer, either fwd or rev, at 1/20 dilution
+*10μL of sample and DH<sub>2</sub>O, such that sample is at 500ng total mass
+Sealed tubes with parafilm.
+Submited Gal-EcR 1, 7, 10, and 12 (the one's with inserts on the digestion gel from 8/4)
+<html><div id="entry"><span id="date"><a name="08-06-2010" class="date">08-06-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Digestions'''
+pACT2
+*1μL Bam
+*1μL Xho
+*6μL pACT2 (tube with a green top)
+*1μL FD Green buffer
+*1μL DH<sub>2</sub>O
+RXRLc
+*1μL Bam
+*1μL Xho
+*9μL RXRLc4
+*2μL FD Green buffer
+*7μL DH<sub>2</sub>O
+Barstar
+*1μL Eco
+*1μL Pst
+*2μL Barstar 3
+*1μL FD Green buffer
+*5μL DH<sub>2</sub>O
-==07-14-2010 [ [[#top|top]] ]==
+Barnase
+*1μL Eco
+*1μL Pst
+*1.5μL Barnase 2
+*1μL FD Green buffer
+*5.5μL DH<sub>2</sub>O
+xGalUAS
+*1μL Spe
+*1μL Pst
+*2μL 5xGalUAS (from Karmella)
+*1μL FD Green buffer
+*5μL DH<sub>2</sub>O
-==07-15-2010 [ [[#top|top]] ]==
+pENT Cup
+*1μL Spe
+*1μL Pst
+*1μL pENT Cup
+*1μL FD Green buffer
+*6μL DH<sub>2</sub>O
+EcR-Gal
+*1μL Xba
+*1μL Pst
+*2μL EcR-Gal 7
+*1μL FD Green buffer
+*5μL DH<sub>2</sub>O
-==07-16-2010 [ [[#top|top]] ]==
+VP16
+*1μL Spe
+*1μL Pst
+*5μL VP16(1)
+*1μL FD Green buffer
+*2μL DH<sub>2</sub>O
+RXRLc
+*1μL Xba
+*1μL Pst
+*9μL RXRLc4
+*2μL FD Green buffer
+*7μL DH<sub>2</sub>O
-==07-19-2010 [ [[#top|top]] ]==
+V24
+*1μL Eco
+*1μL Pst
+*3μL V24
+*1μL FD Green buffer
+*4μL DH<sub>2</sub>O
+V26
+*1μL Eco
+*1μL Pst
+*9μL V26
+*12μL FD Green buffer
+*7μL DH<sub>2</sub>O
-==07-20-2010 [ [[#top|top]] ]==
+PhyB
+*1μL Bam
+*1μL Nco
+*16μL PhyB
+*2μL FD Green buffer
+<html><div id="entry"><span id="date"><a name="08-09-2010" class="date">08-09-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-21-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/0/07/88yescamvexp2.jpg" id="single_image"><img src="http://openwetware.org/images/0/07/88yescamvexp2.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+yes colonies for ligation of exp2 and transformation of camv
-==07-22-2010 [ [[#top|top]] ]==
+'''Digests'''
+<html>
+<div>
+<a href="http://openwetware.org/images/c/ca/86_digests.jpg" id="single_image"><img src="http://openwetware.org/images/c/ca/86_digests.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-==07-23-2010 [ [[#top|top]] ]==
+*ladder
+*1. pACT2 (backbone)
+*2. RxRLC (insert)
+*3. Barstar (insert)
+*4. Barnase (insert)
+*5. 5XGalUAS (backbone)
+*6. PENTcup (backbone)
+*7. V26 (backbone)
+*8. Ecr-Galv (insert)
+****no insert for 2 (RxRLC) or 8 (Ecr-Galv)
-==07-26-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/0/0b/88_other_digests.jpg" id="single_image"><img src="http://openwetware.org/images/0/0b/88_other_digests.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+*ladder
+*RxRLC (Xba and Pst) (insert)
+*RxRLC (Bam and Xho) (insert) NO INSERT HERE
+*PhyB (backbone)
+*V24 (backbone)
+*VP16 (backbone)
-==07-27-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="08-10-2010" class="date">08-10-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+[Image: 810digestslater.jpg]]
-==07-28-2010 [ [[#top|top]] ]==
+*ladder
+*PhyB (backbone)
+*Barstar 4 (insert)
+*V26 (backbone)
+*GalDBD1 (insert)
+*GalDBD5 (insert)
+*B21 (backbone)
+'''Digests 8/10'''
-==07-29-2010 [ [[#top|top]] ]==
+*PhyB with Bam and Nco
+*Barstar with Pst and Eco
+*V26 with Pst and Eco
+*Gal4DBD with Eco and Spe (Gal4DBD minipreps 1 and 5)
+*Arp2BB PCR product with Xba and Pst
+*Arp PCR product with Xba and Pst
+*B21 with Xba and Pst
+*ACC synthase with Eco and Spe
+*Barnase ligation product with Eco and Xba
+<html><div id="entry"><span id="date"><a name="08-11-2010" class="date">08-11-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==07-30-2010 [ [[#top|top]] ]==
+'''colonies from yesterday's transformations/ligations'''
+<html>
+<div>
+<a href="http://openwetware.org/images/2/21/810_colonies_1.jpg" id="single_image"><img src="http://openwetware.org/images/2/21/810_colonies_1.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-==08-02-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/5/51/810_colonies_6.jpg" id="single_image"><img src="http://openwetware.org/images/5/51/810_colonies_6.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html><div id="entry"><span id="date"><a name="08-12-2010" class="date">08-12-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
-==08-03-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/8/8b/Arp2coloniesligb21.jpg" id="single_image"><img src="http://openwetware.org/images/8/8b/Arp2coloniesligb21.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+This picture is not the best picture but we did find colonies on the plate with the arp2/b21 transformation.
-==08-04-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/e/ec/812_5xgaluas.jpg" id="single_image"><img src="http://openwetware.org/images/e/ec/812_5xgaluas.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+transformation from karmella's stock
-==08-05-2010 [ [[#top|top]] ]==
+<html><div id="entry"><span id="date"><a name="08-13-2010" class="date">08-13-2010</a></span> [<a class="top" href="#notebook">top</a>]</div><hr /></html>
+'''Transformations from Yesterday's Ligations'''
-==08-06-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/8/80/VP16Gal4DBD_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/8/80/VP16Gal4DBD_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/f/f6/PENT_cont_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/f/f6/PENT_cont_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-==08-09-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/c/ce/Nost_Act2LacO_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/c/ce/Nost_Act2LacO_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/7/71/Gal4spepst_cont_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/7/71/Gal4spepst_cont_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-==08-10-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/4/40/Gal4_ecoxba_cont_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/4/40/Gal4_ecoxba_cont_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/5/59/Act2Lac_cont_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/5/59/Act2Lac_cont_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-==08-11-2010 [ [[#top|top]] ]==
+<html>
+<div>
+<a href="http://openwetware.org/images/6/6d/Nost_pENT_lig_8-13.jpg" id="single_image"><img src="http://openwetware.org/images/6/6d/Nost_pENT_lig_8-13.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
+<html>
+<div>
+<a href="http://openwetware.org/images/2/2b/EcR-Gal_BB_lig_8-12.jpg" id="single_image"><img src="http://openwetware.org/images/2/2b/EcR-Gal_BB_lig_8-12.jpg" width="240px"/> [click to enlarge]</a>
+</div>
+</html>
-==08-12-2010 [ [[#top|top]] ]==
+Innoculated colonies from EcR-Gal, Nost-pENT, and VP16Gal4DBD. No colonies visible on
+Nost-Act2LacO.
-==08-13-2010 [ [[#top|top]] ]==
+All Controls were clean or had very very few colonies (<2).

Team:Harvard/fences/notebook

From 2010.igem.org

Latest revision as of 02:19, 27 October 2010

notebook