3.4 Leaf Squish & PCR

3.4 Leaf Squish & PCR margaret Wed, 11/16/2011 - 18:38

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?

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

3.4 - Phire Plant kit

3.4 - Phire Plant kit kdorfman Tue, 03/21/2017 - 18:43

Phire 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 - 15:55

M 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)

leaf squish calcs

leaf squish calcs kdorfman Thu, 03/28/2013 - 20:34
Forrxns/pair, & pairs
aliquotmL DEB /pair, & mL total
aliquotmL KOAc/pair, & mL total
aliquotmL isopropyl/pair, & mL total
aliquotmL 70% EtOH/pair, & mL total
aliquotmL T10E5/pair, & mL total
aliquotmL NaOAc/pair, & mL total
aliquotmL 100% EtOH/pair, & mL total
aliquotmL T10E1/pair, & mL total