PCR problem amplifying 4 kb DNA - (Jun/22/2005 )
What kind of method did you choose for the 4kb sized DNA??
I have tried the same cycle condition for the 4kb DNA as my 2kb DNA,but it failed....
So your suggestions are welcomed, I don't know what's problem in the condition.....
A few years back I had to pcr out 2kb and 4kb of Human Acat 2 promoter. The 2 kb worked like a charm, the 4kb was a bit trickier.
Here is what I did: Make sure your final concentration of primer is 0.5uM for each primer, use a final concentration of 300uM for each dNTP, and extension temp of 68C and the denaturation step of 10seconds. Use 0.2 Units of Polymerase.
This above is taken nearly verbatum from Quiagen Taq handbook. I only had to try once to get my 4kb fragment.
according to my experience, there is no anything special to amplify 4kb DNA fragment,extending time should be adapted to 4mins or longer.Best wish!
Maybe lower your extension temperature to 68°C instead of 72°C, depends on which enzyme your using. Also increase your extension time to 4 minutes and increase this with 2 seconds or so for every cycle? I've had no problems amplifying 4 kb, but I started with plasmids.
I think there won t be much difference in amplifying 4 kb length, I feel the extension temperature plays a role. Try to lower the extension temperature.
have a nice day
Thanks everybody, I got successful result at last.....
Yeah, Vairus and pfy1982 , you are both right.
I think the main problem should be the extension time. I did it a bit shorter last time @_@
So I set 5minutes this time, and what I used is Phusion PCR. And I have tested that It is better than Megmix.
pBluescript, I have checked everythink, they are the same as you suggested. Thanks.
Biosaint, The extension temperature I set is 72degree, according to the Phusion kit. how about yours?