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.

Duncan
-- 
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
http://www.dnamp.com
http://www.genesys.demon.co.uk



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