Long PCR

Dr. Duncan Clark duncan at genesys.demon.co.uk
Sat Nov 15 04:58:22 EST 1997

In article <346925EB.153A at mcrcr.med.nyu.edu>, mehboob shivji
<shivjm01 at mcrcr.med.nyu.edu> writes
>I am having a problem amplifying a 2700 bp fragment.
>Anneal time = 2 min @ 55 deg
>Elongation = 3 min @ 72 deg
>Denature = 2 min @ 95 deg
>I would appreciate any suggestions.

Reduce the annealing and denaturation time to 10-30secs. Extension time
is fine.

What are you amplifying from? Is the DNA GC or At rich. If GC rich try
adding DMSO to 5-10% final and or betaine to 1M final. Try using a
Taq/proof-reading pol mix. Should need about 1.25u pol per 50ul PCR. 

If the DNA is extreme AT rich then reduce extension temp to 60 or 62C.

The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288

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