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

Program thermalcycler for the following conditions:

30sec. 92℃

30sec. 40℃

30sec. 50℃

1min. 72℃

repeat cycle 24 times

3.Second round of PCR amplification to incorporate Cy dyes

Prepare the following:

F-100X dNTPs:

10ml 100mM dATP

10ml 100mM dCTP

10ml 100mM dGTP

5ml 100mM dTTP

5ml 1XTE

Reaction tube containing the following for 1-50ml reaction:

15ml PCR product from Step 2

5ml 10X PCR buffer

0.5ml F-100X dNTPs

1ml 50mM Primer 2

1ml 25mM MgCl₂

24ml water

3ml Cy dye (add last)

Program thermalcycler for the following conditions:

30sec. 92℃

30sec. 40℃

30sec. 50℃

1min. 72℃

repeat cycle 24 times

IMMEDIATELY FOLLOWING PCR:

4.Purify probe

Add 400ml 1XTE to Microcon-30 filter and then add Cy3 and Cy5 reactions to the filter.Centrifuge 7min.at max speed.Add 450ml 1XTE to the filter to wash and repeat centrifugation.Repeat wash and centrifugation one more time and then centrifuge filter again for 2min.to remove excess liquid.Invert filter and centrifuge 1min.to collect label.Add 12ml 1XTE,2.55ml 20XSSC and 0.45ml 10%SDS to the colored probe and then denatured by boiling for 2min.Incubate at room temperature for 15min.and then centrifuge at max speed for 10min.Probe is ready for hybridization.