Team:Toronto/Notebook-w07-wed
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Wednesday June 29, 2016
Wednesday, 6/29
Members Present: Hamed, Kat, Cathy, Dk, Tam
LAB:
Morning:
- Made 2 bottles of 500mL of 2×YTPG (for s30 pellets)
- Cultured 100mL of DH10B as part of RbCl competent cell protocol
- PCR amplification of Short Linear LacZ fragment under the following conditions: Normal (being the conditions run previously yesterday, with a shortened annealing time of 20sec), 2 step (with the annealing temperature raised to the extension temperature 72C), 7sec (wherein the extension time was reduced from 15 sec to 7 sec to account for the smaller size of the fragment), and MgCl2 (where the Mg+ concentration was boosted by 0.2uM to account for the present of EDTA)
Afternoon:
- Made rubidium chloride competent cells, stored in -80 as CCD
- Miniprep and nanodrop of previous E.glowli culture:
A | B | C | D | |
1 | sample ID | 260/280 | 260/230 | ng/uL |
2 | Miniorepped E.glowli 1 | -7.31 | 0.09 | 0.8 |
3 | Miniprepped E.glowli 2 | 0.04 | 0 | 0 |
Table1
There appears to be nothing there. Oh well.
- Made 5x glycerol stocks of e.coli DH10B and of RFP containing DH10B. made 3x glycerol stocks of E.glowli containing E.coli.
- Nanodrop of the PCR results.
A | B | C | D | |
1 | Sample name | 260/280 | 260/230 | Concentration (ng/uL) |
2 | MgCl2 | 1.59 | 0.96 | 912.9 |
3 | 2 steps | 1.3 | 0.62 | 344.8 |
4 | Regular | 1.53 | 0.84 | 704.5 |
5 | 7 sec | 1.37 | 0.74 | 4096 |
Table2
Nanodrop results post PCR of the Short Linear LacZ fragment. No PCR purification beforehand.
- Ran a gel along with previous PCRs of the LacZ.
GEL ELECTROPHORESIS: Lane 1: Sample with increased MgCl2 concentration and annealing time at 20sec instead of 30sec. Lane 2: 2 Step PCR of LacZ, with the annealing temperature equivalent to the extension temperature. Lane 3: Regular PCR with an annealing time of 20 sec instead of 30 sec. Lane 4: 3kb ladder. Lane 5: LacZ PCR with extension time of 7 sec instead of 15 sec. Lane 6: PCR from previous day at 70.2C with an annealing time of 30 sec. Lane 7: PCR from previous day at 69.1C with an annealing time of 30sec. Lane 8: PCR from previous day with additional MgCl2 and a annealing time of 30sec.
- Appears that running a 2 step PCR might resolve the issue of our PCR insters concatemerizing.
- 5ml Overnight culture of E.glowli and the single colony from L1 plates.
- Made 10% w/w of L-arabinose solution for inducing the PBAD promoter found in E.glowli.
Administrative:
TO DO:
For the next day:
LAB TEAM:
LAB MANAGERS:
Gmail correspondence:
Meetings/Notes:
References:
