goa at aber.ac.uk
Fri Feb 9 06:08:27 EST 1996
In article <4fcphn$1nt at news.uni-c.dk>, voldborg at biobase.dk says...
>Hello out there,
>I am trying to make a PCR fragment of aprox. 5.5kb, and introducing a
restriction site in both ends of it. The 5'end is highly GC rich, and the
>e 5' primer and CG pairs for the 3' primer.
>I perform the PCR with TaqPlus DNA polymerase from stratagene, using
elongations cycles from 6 to 10 min, but everytime all I get is a smear,
and not one
>Does anybody know what I am doing wrong, any help will be apreciated
>voldborg at biobase.dk
Check your primer sequences and try to get their tms balanced (2C-A/T,
4C-G/C), Ensure that they are not complementary at their 3'ends, I usually
just do this by eye.
Add about 25pmoles of each to the reaction, I usually run 50ul volumes.
Set up a magnesium titration from 1.0mM-3.0mM.
Hot start by heating to 95C for about 3mins and adding 0.5ul Taq under the
oil, convection mixes it in.
Do maybe 25 cycles of : 30s at 5C less than tm/ 1min at 72C/ 30s at 94C.
If you still get a smear in all lanes the up the annealing temp to just
below the predicted tm and retitrate.
If you get little to no product either drop the annealing temp, increase
the annealing time or decrease the ramp rate.
Hope that this helps. Gordon.
More information about the Methods