PFU PCR Amplification Problems

[admin]

I got this question from a new user:

i am working on an amplification with the pfu and unfortunately it does not work and i do not really know what to do because i have tried in different ways to change the conditions of the reaction but it always gave negative results. please do help me to find a solution.

if you have ever worked with pfu tell me the conditions of the experiment to see if they are the same as the ones i am using.

Can anyone help here?
thanks

[̑̑̑]

yo, i have worked with pfu to amplify when i want fidelity. The last exp i have done is amplifying a chimeric construct of a drosophila gene fused to egfp.

This is the way i have done it:

pcr conditions: 55C 30 cycles 1minute extension (this could vary depending on template to be sequenced, primers Tm and anneling T and so on)

pcr cocktail:
2 ul template (from a klenow fragment reaction)
2ul 10x pfu buffer
2ul primer f (20nM)
2ul primer r (20nM)
0.2 ul 20mM dNTPs
0.2 ul cloned pfu pol (stratgene)
0.1 ul taq pol
mq water to to 20ul

pcr run on applied biosystems GeneAmp® PCR System 9700 (but it should be ok on other machines)

it actually did work very well for me, nice fatty band when ran on gel (in fact i took it down to 20 cycles) and ultra high fidelity when sequenced.

Hope it helps