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Request for information on helicase dependent amplification

HDA PCR

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#1 Prasadiit

Prasadiit

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Posted 10 September 2013 - 04:34 AM

Hello,

I am trying to amplify a 120bp amplicon from a plasmid using the Isoamp II
Helicase Dependent Amplification (HDA) kit from New ENgland Biolabs. The 
primers I have designed are 25bp and 28bp respectively and have a T_m 
(melting temperature) of 80 deg.C.

I can get the PCR to work with these primers without any problem, but am 
not able to make the HDA kit work. My work needs me to come up with a
single-temperature amplification protocol.

I have tested the kit with the template DNA supplied with it- the kit
works fine. I have tried HDA at 60, 65, 70, 75 deg.C without any 
success. I am not sure if the helicase enzyme can withstand a higher 
HDA temperature, say, 75 deg.C. 

If anyone on this forum has experience with optimizing HDA, could
you please help? Should I redesign my primers or there is a way
to make these work in HDA?

I would appreciate any feedback that you might be able to give me.
I'll also be happy to share sequences etc. if needed.

Many thanks in advance.

Regards,
Prasad






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