Team:Harvard/fences/notebook

(Difference between revisions)

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	06-25-2010
Week 3	06-28-2010	06-29-2010	06-30-2010	07-01-2010	07-02-2010
Week 4	07-05-2010	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	07-19-2010	07-20-2010	07-21-2010	07-22-2010	07-23-2010
Week 7	07-26-2010	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 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, 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.

300px 300px 300px

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

500px

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

• 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 ∞

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

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: 350px

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

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

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

350px

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

350px

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

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.

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

350px

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.

File:71barnasepcrweird.jpg

07-05-2010 [ top ]

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

File:76B21 digest gel.pdf

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].

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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

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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 ]

File:78PCRgelGalBarnaseLac.pdf

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.

File:78gelextractskeleton.pdf

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"

Saturday 7/11

Innoculating colonies from Friday's ligations

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

Sunday 7/12

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:

File:07122010ve3ctordigestmirbraz2.jpg

Sequencing

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

07-13-2010 [ top ]

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

07-19-2010 [ top ]

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

07-20-2010 [ top ]

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

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

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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

240 px FINALLY! EXP2 PROMOTER AMPLIFIED!

~1000kb

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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.

07-26-2010 [ top ]

Successful ACC synthase transformation:

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