3.4 Leaf Squish & PCR
3.4 Leaf Squish & PCR margaret Wed, 11/16/2011 - 18:38Salk 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
3.4 - Phire Plant kit
3.4 - Phire Plant kit kdorfman Tue, 03/21/2017 - 18:43Phire Plant Direct PCR Kit
Fisher F-130WH
Per plant sample:
- 20 uL Dilution Buffer
Per 20 uL reaction:
- 10 uL 2X Phire Plant PCR buffer
- (primers to 0.5 uM)
- 0.4 uL Phire hot start polymerase
- 0.5 uL Plant squish supernate
- sterile water to make 20 uL
Lab 3.4 2012
Lab 3.4 2012 kdorfman Tue, 03/26/2013 - 15:55M 3/26/12
CORRECT THE VOLUMES IN THE LAB MANUAL 21 mL 10X in 210 mL total!
Goal for this lab: Genotype Salk line plants, looking for homozygous mutants.
Use 3 primers in a single reaction (GSP1, GSP2, LBP)
LB 195 LBb1.3 ATTTTGCCGATTTCGGAAC (111 bp from left border)
Materials
- PCR strip tubes
- Harris cutting mat
- Harris Uni-Core (0.5 mm) Cutting Tool
- Beakers for bleach
Reagents
- 2% bleach (12 mL bleach + 588 mL dH2O)
- 5U/µL Taq (3.5 µL per pair)
- 10X polymerase buffer (21 + 21 = 42 µL per pair)
- 2.5 mM dNTP mix (51 µL per pair)
- Sterile dH2O
- Working stocks of primers, including LBP diluted to 10 µM (17µL/rxn)
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?