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Team:UCL/Labbook/Week11
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Revision as of 21:48, 4 October 2013
Lab Weeks
Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18
Week 11
Bacterial Lab
Monday 12th August
Chloramphenicol was produced and stored at -20°C. In order to produce glycerol stocks, pSecTag2A and pSB1C3 were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37C o/n.
Tuesday 13th August
Results from the following plates:
| Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
|---|---|---|---|
| pSecTag2A Cells | Yes | Yes | 100+ |
| W3110 Cells Amp | Yes | No | 0 |
| pSecTag +ve control | No | Yes | 100+ |
| PSB1C3 Cells | Yes | Yes | 0 |
| W3110 Cells Chlor | Yes | No | 25 |
| PSB1C3 +ve control | No | Yes | 0 |
This indicated that the pSecTag2A cells are acceptable to use for glycerol stock generation and for plasmid purification. Miniprep was performed on the two incubated Falcon tubes from yesterday.
The results concerning pSB1C3 indicated that the chloramphenicol did not work, and the glycerol stock is dead. Therefore a new glycerol stock was sought after and chloramphenicol was remade.
Ampicillin & chloramphenicol were remade and pSB1C3 was located in the iGEM 2012 distribution kit. Ampicillin & chloramphenicol were both tested by producing 1x positive and 1x negative plate for each antibiotic streaked with W3110 cells. Left to incubate at 37°C overnight.
Glycerol stocks of pSecTag2A was grown, and a miniprep was carried out. PCR could not be carried out as the primers for pSecTag2A had not arrived.
Wednesday 14th August
Two analytical digests of pSecTag2A with EcoR1-HF and Dpn1 were carried out.
| Item A | Volume A (ul) | Item B | Volume B (ul) |
|---|---|---|---|
| pSecTag2A | 5 | pSecTag2A | 5 |
| EcoR1-HF | 1 | Dpn1 | 1 |
| Buffer 4 | 1 | Buffer 4 | 1 |
| BSA | 0.5 | BSA | 0.5 |
| dH20 | 2.5 | dH20 | 2.5 |
| Total | 10 | Total | 10 |
[insert image of gel]
Told by supervisor Dr Darren Nesbeth to use a ratio of 3:1 inoculum:80% glycerol when making glycerol stocks.
Darren's instructions:
pSB1C3
Transfer entire glycerol tube contents to 10mL LB ND in a 50mL Falcon & measure OD. Place in 37C shakee overnight. Measure OD next morning. If there is growth in the inoculum use a loop to streak onto chloramphenicol and ND plates again.
pSecTag2A
50ul of 2013 pSecTag2A glycerol stock to inoculate 10mL LB ampicillin in a 50mL Falcon. Grow overnight then generate 15x glycerol stocks in 1.5mL eppendorfs. Store at -20°C.
Thursday 15th August
OD results for pSB1C3 LB ND inoculum
| Plasmid | OD Before | OD After | ||||
|---|---|---|---|---|---|---|
| PSB1C3 (LB ND) | 0.052 | 0.5 |
| ul | EcoR1 single digest | Spe1 single digest | Double digest | Uncut |
|---|---|---|---|---|
| pSecTag2A | 5 | 5 | 5 | 5 |
| EcoR1 | 1 | 0 | 1 | 0 |
| Spe1 | 0 | 1 | 1 | 0 |
| BSA | 0.5 | 0.5 | 0.5 | 0.5 |
| Buffer 4 | 1 | 1 | 1 | 1 |
| dH20 | 2.5 | 2.5 | 1.5 | 3.5 |
| Total | 10 | 10 | 10 | 10 |
iRRE+PC+RBS and PC+RBS (containing pSB1C3, taken from 2012 iGEM boxes) were plated onto cmp plates. Incubated at 37°C overnight.
Two falcons for each stocks, one containing 5ul and the other 15ul, both were inoculated in 2ml LB+2ml chloramphenicol. Incushaker at 37°C overnight.
Friday 16th August
Falcons were retrieved:
RESULTS
Inoculum:
| Falcon contents (+LB+Amp) ul | Colony growth | Absorbance |
|---|---|---|
| IRRE+PC+RBS (15ul) | Yes | 0.8 |
| IRRE+PC+RBS (5ul) | Yes | 0.7 |
| PC+RBS (15ul)/td> | No | 0.06 |
| PC+RBS (5ul) | No | 0.08 |
Plates
| Plate contents (+LB+Amp) ul | Colony growth | Absorbance |
|---|---|---|
| IRRE+PC+RBS | Yes | 100+ |
| IRRE+PC+RBS | No | 100+ |
| PC+RBS/td> | Yes | 0 |
| PC+RBS | No | 20 |
The PC+RBS plates & falcons were discarded
IRRE+PC+RBS (15ul inoculum) -> underwent miniprep
IRRE+PC+RBS (5ul inoculum) -> 4x glycerols were made: 500ul culture + 166ul 80% glycerol, stored in MMP -20C iGEM 2013 box.
pSB1C3 Gel sd: EcoR1 & Pst1 + dd
| Item (ul) | EcoR1 | Pst1 | Double digest | Uncut |
|---|---|---|---|---|
| pSB1C3 | 5 | 5 | 5 | 5 |
| EcoR1 | 1 | 0 | 1 | 0 |
| Pst1 | 0 | 1 | 1 | 0 |
| Buffer 3 | 1 | 1 | 1 | 1 |
| BSA | 0.5 | 0.5 | 0.5 | 0.5 |
| dH20 | 2.5 | 2.5 | 1.5 | 4.5 |
| Total | 10 | 10 | 10 | 10 |
3ul loading dye to each solution
Lanes:
2 - Hyperladder, 4 - EcoRI, 5 - PstI, 6 - dd, 7 - Uncut
Results from gel: 1kb DNA ladder showed on gel. No bands for EcoR1, Pst1, DD and Uncut plasmid were seen - indicating that no DNA was present -> possibly due to problems with the miniprep.
Therefore from the iGEM 2012 plate 5 (distribution kit) well 23.O, DNA containing BBa_j04450 (colonies are clearly red in colour - RFP) in pSB1C3 was located. The dried DNA was resuspended in 10ul dH2O, left to sit for 5 minutes, then transformed into competent cells -> streaked onto plates and left to incubate at 30°C over the weekend.
Mammalian Lab
Monday 12th August
HeLa cells have confluency of about 60%. Passaged the HeLa cells and ‘backup’ HeLa cells. We set up two 6-well plates at 0.25 x 10^6 cells/ml in preparation for Zeocin kill curve. Cell count involved (0.45 cells/ml).
Tuesday 13th August
Prepared media with various concentrations of zeocin for each of 6 wells.
| Concentration of zeocin (µg/ml) | Volume of zeocin (ml) | Volume of DMEM + 10% FBS + 2 mM L-Glu (ml) |
|---|---|---|
| 0 | 0 | 30.0 |
| 50 | 15 | 30.0 |
| 100 | 30 | 30.0 |
| 250 | 75 | 29.9 |
| 500 | 150 | 29.9 |
| 1000 | 300 | 29.7 |
T75 flasks: - 90% confluency T25 flasks - 30% confluency 6-well plates - 30% confluency
Wednesday 14th August
T25 flasks 90% confluency
Disc 1 Disc 2
| Disc 1 | Disc 2 | Concentration of zeocin (µg/ml) | Confluency (%) | Cell Appearance | Comment | Confluency (%) | Cell Appearance | Comment |
|---|---|---|---|---|---|---|
| 0 | 65 | Healthy | Healthy, few swell | 65 | Healthy | minimal floaters |
| 50 | 40 | Healthy, few swell | few floaters | 65 | occasional swell | moderate floaters |
| 100 | 40 | Half/moderate swell | Moderate floaters, minor infection | 50 | 40% swelling | minimal floaters |
| 250 | 25 | most/moderate swell | many floaters | 60 | moderate/severe swelling | many floaters |
| 500 | 40 | most clumps dead/ swell | many floaters, possibly infection | 65 | severe swelling all over | large number of floaters |
| 1000 | 45 | very severe death/ swell | many floaters, minor infection | 60 | severe swelling all over | moderate number of floaters |
The two T25 flasks with revived HeLa have confluency of about 90%; T75 flask with ‘backup’ cells 70% confluent. The T25 flasks are discarded.
Friday 16th August
| Disc 1 | Disc 2 | Concentration of zeocin (µg/ml) | Confluency (%) | Cell Appearance | Floaters | Confluency (%) | Cell Appearance | Floaters |
|---|---|---|---|---|---|---|
| 0 | 90 | Healthy | Minimal | 95 | Healthy | minimal |
| 50 | 80 | minor swell | moderate | 90 | minor swell | moderate |
| 100 | 65 | Minor swell, minor death | Many | 75 | Minor swell, minor death | moderate |
| 250 | 50 | severe swelling, death | many | 50 | severe swelling, death | many |
| 500 | 60 | severe swelling, death | many | 50 | severe swelling, death | many |
| 1000 | 40 | severe swelling, death | many | 50 | severe swelling, death | many |
Split and passaged stock HeLa into 2 flasks
Saturday 17th August
Passaged HeLa cells
