Difference between revisions of "Team:Toronto/Notebook-w06-thu"
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Latest revision as of 06:57, 19 October 2016
console.js:26
Thursday June 23, 2016
Thursday, 6/23
Members Present: Marc, Andrea, Celine, Davesh, Karim, Kat
LAB:
Morning:
- Transformation did not work - the colony counts are not consistent across different concentrations and duplicates. As there are no controls (ie, with different batches of competent cells, or the cells plated on just LB) it is difficult to assess wether the issue is with the competency of the cells, the transformation, or the viability of the cells themselves. Therefore another attempt with controls was done for the following day.
- Made LB + CAM Plates
- Miniprepped RFP:
A | B | C | |
1 | RFP B1 | RFP B2 | |
2 | 260/280: | 1.54 | 1.6 |
3 | 260/230: | 0.72 | 0.69 |
4 | ng/uL: | 10 | 13.4 |
Table3
Afternoon:
- Transformation with the following conditions (including controls):
A | B | C | D | E | |
1 | 0.5pg | 10pg | 50pg | RFP 1A | Control no plasmid |
2 | CCA | CCA | CCA | CCA | CCA |
3 | CCB | CCB | CCB | CCB | CCB |
4 | CCC | CCC | CCC | CCC | CCC (also plated on LB) |
Table1
Table 1: Set up for a comparative transformation between different batches of competent cells. CCA and CCB are the first two batches of MgCl chemically competent cells. The third batch, CCC, was made through the use of RbCl.
●
Gel electrophoresis of both EcoRI and PstI digested RFP plasmid, with and without DpnI. Including a 3kb ladder and miniprepped RFP.
GEL - LANE 1: RFP plasmid digested with EcoRI and PstI. Fragments correspond to a backbone at around 2kb and insert at around 1100bp. LANE 2: RFP plasmid digested with EcoRI, PstI, and DpnI. Largest DpnI fragment can be observed at around 750bp. The insert is 130bp shorter than the full size (976 v.s. 1106[including overhangs]) due to a DpnI restriction site within it. The LANE 3: 3kb ladder. LANE 4 + 5. Miniprepped RFP (3139bp) . Nonspecific banding most likely due to supercoiling due to the circular nature of the plasmid.
●
Concluded that our restriction enzymes work appropriately.
Administrative:
TO DO:
For the next day:
If transformation works:
-
A | B | C | |
1 | Check reagents | Check reagents | |
2 | Resuspend Short Linear LacZ-alpha | ||
3 | Resuspend Backbone primer fwd, Backbone primer rev | Resuspend UNS1 rvs primer and UNS8 primer fwd | 20 min total |
4 | Check lab notebook for optimal PCR temp. | ||
5 | PCR | PCR | 1.5 hr |
6 | PCR Purification | PCR Purification | 1.5hr |
7 | Gel electrophoresis | Gel electrophoresis | 1.5hr |
8 | Store at 2C | Store at 2C | |
Table2
If transformation doesn't:
- make more rubidium chloride competent cells
LAB TEAM:
LAB MANAGERS:
Gmail correspondence:
Meetings/Notes:
References:
