Koeris/Notebook/2007-1-18: Difference between revisions
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*Loading volume: 20ul of PCR Rx = 40% product | *Loading volume: 20ul of PCR Rx = 40% product | ||
*1% agarose TAE gel, ran @ 100V for 60min | *1% agarose TAE gel, ran @ 100V for 60min | ||
[[Image:2007-01-18 new primers + control.tif|thumb|Description]] | |||
Revision as of 17:59, 18 January 2007
Transformations
Continued from 1/17. A construct didn't give any colonies -> try using more plasmid DNA, i.e. not 1ul but 2.5ul and 5ul in two aliquots.
Also plate ON RT aliquot, maybe there is one or two transformants, though historically there is a lower chance of that happening.
P. aeruginosa mer operon cloning
Continued from 1/15.
Approach
I designed two primers: one new set of mer operon primers, that are significantly longer than before (merSO 2F&R 44bp, 36bp), as well as a set of check primers to see if the DNA preparation is usable at all, or if it is degraded.
I will use the Invitrogen Taq SuperMix for the PCR.
PCR set-up
- 50 ul total reaction volume
- 1ul template DNA @ 300ng/ul
- 200pmol primer mix or 400pmol
- 47ul Taq SuperMix
Pipetting scheme
Labels in the respective field codes
| Primer | Tuber Label |
|---|---|
| merSO 2F&R 200pmol | A1 |
| merSO 2F&R 400pmol | A2 |
| recF 1F&R 200pmol | A3 |
| recF 1F&R 400pmol | A4 |
Thermal profile
- 92 deg C - 5min
- 92 deg C - 30s
- 50 deg C - 45s
- 72 deg C - 120s
- Cycle back to 2. 29X
- 72 deg C - 10min
- 4 deg C - indefinite
Gel Run
IMPORTANT: Load genomic DNA on the gel as well to test -> at least 1ug - 2ug
- 14-comb gel @ 50ml
- Loading volume: 20ul of PCR Rx = 40% product
- 1% agarose TAE gel, ran @ 100V for 60min
