Amplification and Labelling with Cy dyes
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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~Eadd_reaction_mix_1~J~K~Hp~M~2~1~Kp~M2nd_round~Eadd_reaction_mix_2~K~Hp~M~2~1~Kp~M8min.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.
