Amplification and Labelling with Cy dyes

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