Each embryo or yolk sac is digested O/N at 50-55°C in 100 µl of PCR Lysis Buffer containing 100 µg/ml proteinase K (either with continuous gentle agitation, or with a brief vortex in the morning followed by 1-2 hours more digestion). Digested samples can be stored at room temperature.
PCR Lysis Buffer (aliquots containing ProK can be stored at -20°C)
50 mM KCl
10 mM Tris pH 8.3
2 mM MgCl2
0.1 mg/ml gelatin
0.45% NP-40
0.45% Tween-20
Use 4 µl of each DNA sample with 8 µl of water in each PCR reaction. Denature at 95°C for 10 minutes in the PCR machine. At 85°C add 8 µl of the cocktail (freshly made and premixed; can be added through the oil.)
|
Cocktail |
per reaction |
|
100 ng/µl primer 1 |
1.2 µl |
|
100 ng/µl primer 2 |
1.2 µl |
|
100 ng/µl primer 3 |
1.2 µl |
|
10X PCR buffer |
2.0 µl |
|
12.5 mM MgCl2 |
2.0 µl |
|
10 mM dNTPs |
0.4 µl |
|
5 U/µl Taq |
0.2 µl |
Cycle conditions
|
94°C |
1 min |
|
62°C |
2 min |
|
72°C |
4 min with 3 second autoextend |
|
40 cycles |
Run the whole sample on an agarose gel with markers, a no DNA negative control, and positive controls. The primers are in massive excess and will make a low molecular weight smear on the gel. The most typical problem with the procedure is a failure of the reaction due to too much DNA, especially if tissue is taken from a big tail cut etc. This results in a DNA smear from 100 bp to several kb.
A Conlon Lab Protocol
pdf file for this protocol