PCR GENOTYPING PROTOCOL - WhiteLab
Stock
Solutions
10X
Modified Gitschier Buffer (10XMGB)
500 ml
2 M Tris, pH 8.8 3.35 ml
1 M (NH4)2SO4 1.66 ml
0.5 M MgCl2 1.34 ml
0.5%
gelatin 2.0 ml
autoclaved H2O 1.65ml
10.0 ml
10X
GB =
10X MGB without gelatin
500 ml
2 M Tris,
pH 8.8 167.5 ml
1 M (NH4)2SO4 83.0 ml
0.5 M MgCl2 67.0 ml
autoclaved H2O 182.5 ml
Digestion buffer = 1X
GB, 1% b-mercaptoethanol, 0.5%
Triton X-100
20 ml 10X GB 2.0
ml
b-mercaptoethanol 0.2 ml
Triton X-100 0.1 ml
water 17.7 ml
50 ml
10X GB 5.0 ml
b-mercaptoethanol 0.5 ml
Triton X-100 0.25 ml
water 44.25 ml
100 ml
10X GB 10.0 ml
b-mercaptoethanol 1.0 ml
Triton X-100 0.5 ml
water 88.5 ml
Genomic DNA Digestion & Preparation
1. digest tail piece in 50 ml of digestion buffer
2. heat-inactivate in 95ēC for 10 minutes
3. add proteinase K at a final concentration of
1 mg/ml (2.5 ml of 20mg/ml stock)
4. incubate at 55ēC for 1 hour
5. mix
6. incubate at 55ēC for 1 hour
7. heat-denature the proteinase K at 95ēC for 10
minutes
PCR Protocol
Pre-Mix
RedTaq buffer 2.0 ml
DMSO 2.5
ml
primer 1 2.5
ml
primer 2 2.5
ml
H2O 9.5 ml
1. make pre-mix for correct number of samples (+
a few extra) and add 19 ml to each tube
2. add 1 ml of sample DNA to each tube
dNTP Mix
dNTP working stock 1.0 ml
RedTaq buffer 0.5 ml
H2O 1.5 ml
3. make appropriate amount of dNTP Mix and keep
on ice
4. place tubes with Pre-Mix in thermal cycler
for 3 minutes at 95ēC
(program <MAIN>/DENATURE)
Taq
RedTaq 2.0
ml
5. add RedTaq to dNTP Mix on ice
6. after 3 minutes in thermal cycler, add 5 ml of Taq/dNTP mix to each tube
7. continue PCR program as follows (currently
program <MAIN>/TLF)
PCR Profile
95ēC 3
minutes
add Taq/dNTP mix
93ēC 30 seconds
56ēC 30
seconds 30 cycles
65ēC 2 minutes,
30 seconds
72ēC 5
minutes