Salk genotyping: Leaf Squish & PCR
See revised instructions handout below.
CORRECT PCR CALCS IN HANDOUT
correct PCR cycle. should be
- 98C 15s
- 60C 30s
- 72C 1min 32-40 cycles
Goal for this lab: Genotype Salk line plants, looking for homozygous mutants.
the RP is always on the side of the flanking sequence, that is, RP is always on the 3' end of the insertion. Therefore, the PCR reaction should always be set up as LB+RP for HM and LP+RP for WT. http://signal.salk.edu/tdnaprimers.2.html
Use 3 primers in a single reaction (GSP1, GSP2, LBP) OR
Run 2 separate gels
LB 195
LBb1.3 ATTTTGCCGATTTCGGAAC (111 bp from left border)
Should be on a long-lab day! If it can't be on the long day, it should stop at DNA extraction, then do PCR plus gel the next period!
Set out the 24-tube microcentrifuges. They have 13 samples.
Materials
(See materials for Lab 1.1)
| ingredient | mL per rxn | x 26 rxn | x 19 pairs |
|---|---|---|---|
| DEB | 0.6 | 15.6 | 296.4 |
| KOAc | 0.25 | 6.5 | 123.5 |
| Isoprop | 0.6 | 15.6 | 296.4 |
| 70% EtOH | 1.4 | 36.4 | 691.6 |
| T10E5 | 0.225 | 5.85 | 111.15 |
| NaOAc | 0.025 | 0.65 | 12.35 |
| 100%EtOH | 0.5 | 13 | 247 |
| T10E1 | 0.05 | 1.3 | 24.7 |
Put out 3 double block dry baths at 65C
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DNA yield from leaf punch
The yield is low - many student get no bands at all. Not much better than when we used the older quick squish buffer.
One pair repeated the experiment with quick squish and did no better.
Check primers first?