Melbourne/Lab Notebook gv 1
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June 2007: Create Glycerol stocks of gas vesicle plasmids and cells supplied by Maura Cannon
- Make LB
- Make TE
- Transformation of plasmids provided on papper:
- Soak one of the two filter papper strips provided by Maura Cannon in 40uL of TE (10mM Tris 0.1mM EDTA)
- Use 1uL of this to Transform DH5a super competant cells, and plate onto LB Agar Plates Amp (100ug/mL) overnight.
- Transformation protocol produced only 3 colonies of pNL26(12kbp) but many more pNL29(6Kbp)
- Control(no plasmid) plates were clean.
- Pick colonies from Plates and culture overnight . (pNL26T1..3 & pNL29T1..3)
- Grow Colonies of cells containing plasmid growing on Agar.
- Streaked out coloies from Agar provided by Maura pNL26 and pNL29
- Pick colonies from Plates and culture overnight (pNL26P1..3 & pNL29P1..3)
- Produced glycerol stocks from 900uL of each overnight culture.
- Minipreped remaining culture (4mL) giving 100uL.
- Measure DNA concentrations using 2uL of miniprep(100uL) diluted in 98uL of TE.
| Colony Sample no: | 1 | 2 | 3 |
|---|---|---|---|
| pNL26P (from Agar) | 20 | 31 | 33 |
| pNL26T (from plasmid) | 29 | 29 | 31 |
| pNL29P (from Agar) | 28 | 27 | 28 |
| pNL29T (from plasmid) | 36 | 35 | 31 |
- Digest 26uL of each in Buffer2 with 1uL HindIII (20U) at 37degC for 1h35min. (30uL reaction)
- Prepared 20 lane 100mL agarose gel
- Loaded 20uL of digest samples in the following lane order
- All pNL29 (8983bp) minipreps were cut into 3 fragments as expected (4852bp,3479bp,652bp)
- All pNL26 (10318bp) minipreps were cut into 4 fragments as expected (4852bp,3479bp,1335bp,652bp)
- See Gell
- Performed serial trippling dilution of 1Kb+ marker to determine sensitivity of Ethidium bromide visualisation on Gel. Based on 1650bp band (76ng in 20uL of marker) we can see about 10ng of Dna. Hence to see 652bp band in pNL26(10318bp) we needed to digest at leat 10ng x 10318/652 /20ng/ul=8uL of minipreped pasmid , so we used 26uL above.
- Due to low yeild Craig performed a second overnight culteure and used Alkaline Lysis miniprep protocol to obtain 10.9mg/ml pNL29 and 9.9mg/ml pNL26, however these were not used.
