1.Two rounds of DNA synthesis to incorporate fixed sequences
Prepare the following:
Reaction tube containing the following:
7ml DNA to be amplified and labelled
2ml 5X T7 Sequenase Buffer
1ml 50mM Pimer 1 (GTTTCCCAGTCACGATCNNNNNNNNN)
Reaction mix 1 (for 2 reactions):
2ml 5X T7 Sequenase buffer
3ml 10mM dNTP mix
1.5ml 0.1M DTT
3ml 500mg/ml BSA
0.6ml 13U/ml T7 Sequenase
Reaction mix 2 (for 2 reactions):
1.4ml Sequenase dilution buffer
0.6ml 13U/ml T7 Sequenase
Program themalcycler for the following conditions:
2min. 94℃
2min. 8℃ *1st round,add reaction mix 1;
2nd round,add reaction mix 2
8min.ramp to 37℃
8min. 37℃
repeat cycle once
Dilute products with 35ml 1XTE.
2.First round of PCR amplification with fixed sequence primer
Prepare the following:
Reaction tube containing the following for 1-100ml reaction:
15ml diluted products from Step 1
10ml 10X PCR buffer
2.5ml 10mM dNTPs
2.5ml 50mM Primer 2 (GTTTCCCAGTCACGATC)
1ml 5U/ml Taq (QIAGEN)
2ml 25mM MgCl2
67ml water
