Assembly PCR problem - (Oct/29/2007 )
Hello,
I am trying to build a 300 bp insert using assembly PCR. I am using 14 oligos about 40 nucleotides each for my assembly reaction and two flanking primers 14 and 25 nucleotides each for my amplification reaction. The Tm for both reactions is about 55-56 C, so I varied my annealing temperature between 52 and 54 C. The first reaction conditions were as follows: 300 nM oligos, 0.2 mM dNTPs, 1X Thermopol Buffer (20 mM TrisHCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1 % Triton X-100), 3 units of VentR polymerase. The first reaction protocol was eight cycles of: 94 C (1.5 min), 52 C (2 min) and 72 C (3 min). The conditions for the second reaction were the same as the first one except, 2 microlitres of the first PCR reaction were used as a template for the second reaction (flanking primer concentration was 300 nM). The second reaction protocol was 25 cycles of: 94 C (30 sec), 52 C (2 min) and 72 C (1.5 min).
My problem is that I am not obtaining my desired 300 bp product after the second reaction. I do get a smear on a 1% agarose after my first reaction which is expected in the first stage of assembly PCR but I see nothing on the second reaction lane. I tried to modify the protocols by increasing the melting and annealing temperatures and times and also increasing the extension time. I also tried doubling the number of cycles of the first reaction (to 16) and increasing the number of cycles in the second reaction to 30. Still, I could not obtain my desired product.
Thank you in advance,
Alex.
I've never used the technique before Alex but it seems the most likely explanation is that either the 300 bp template is not present in the 2nd reaction (the 14 oligos in the 1st reaction are not annealing to one another), or the outside primers are not working in the 2nd reaction.
One thing you could do instead of using all 14 oligos together in the 1st reaction is to setup two individual reactions containing 7 oligos from each half of the insert and then in your second reaction amplify each half of the insert and join the two halves together afterwards. It's a little more work but it may help you diagnose which primers are giving you grief and at the same time allow you to make some progress with making your construct.
Seeya, Rob
Hi I have tried the same Assembly PCR but i did it with
1) each reaction contains only 2 primers ( in ur case 7 reactions),
2) using very low anneling temp. in our case 40 degree and 30 cycles.
3) 5uM of each primer is required and the PCR buffer is similar to what a normal PCR (i.e. 2mM MgCl2 and etc).
And we combine each of the set together for second PCR ( we run gel to make sure they are there) or what u can do is to do similar combination of those step 1) again.
The final PCR which consist of 10 cycles of Tm at 40 degree and 20 cycles of your desire TM (55-56 in your case).
We have success on everyone fragment that we get. This method may require more work but you can also complete it in 2-3 days with less failure. I hope this can be help.
Joseph
As far as i understood vent polymerase produces blunt ends not the 3' overhangs as taq.
Might be this is the problem why u are not getting ur product in second PCR.
Just look into it.