"
Team:Harvard/fences/notebook
From 2010.igem.org
notebook calendar
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.
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 |
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
