LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION
- 点击： 作者： 来源： 日期：2007-04-03 本站论坛
PCR
1. Aliquot master mix 2 / each individual DNA template first. Keep on ice.
2. Immediately prior to amplification step add appropriate volume of mix 1. Spin briefly and proceed to PCR step immediately.
3. Carry out PCR as follows:
TECHNE PROGENE
Cycling conditions:
Initial denaturing 94 C, 2 min no. of cycles
94 C, 10s - 65 C, 30s - 68 C, 12 min x10
94 C, 10s - 65 C, 30s - 68 C, 12 min + 20s per cycle x20
Final extension 72oC, 5 min - Refrigerate
Cycling parameters/PCR machine used can affect the success of this protocol. See the additional notes at the end of this document for guidance.
4. Store samples at 4oC prior to digestion.
AGAROSE GEL ELECTROPHORESIS
1. Prepare a 0.6% agarose gel:
0.48g agarose
80 ml 0.6 x TBE
0.5 µg/ml Ethidium bromide
