Condition for amplifying >6k PCR - Long PCR amplification (Oct/11/2005 )
Hi, I'm currently doing an amplification of 6kbp PCR.
What is the recommended PCR condition for doing it?
I'll be using CLontech BD advantage Genomic Polymerase.
Thanks a lot!
As for any PCR, read the manual, make sure elongation is long enough (and probably should be done @ 68°C), make sure your primers are compatible (Tm-wise) and not forming dimers, start with standard Mg-concentration, have an annealing temp. of 5°C < Tm and see what you get.
I did PCR for approx 5KB fragement and used Expand Long PCR kit from Roche. There were 3 different buffers with differing MgCl2 concentrations and other characeterestics. I was not successful with Buffer 1 but Buffer 2 yielded a band. I used genomic DNA to amplify my sequence.
The PCR conditions i Used were
step 1
94C for 2 min
Cycle starts
94C 30sec
68C 5 sec (primer Tm 72)
72 5 min
35 cycles
Yeah I agree with Vairus..It depends on which kit or polymerase one is using.
How much DNA template do i need?
What do I have to take note if I'm going to use DNA extracted from tissue samples?
I tried the PCR, but it keeps giving me lower bands ~ 300bp.
I have already lowered my [primers] to 0.4pmol/ul
The PCR conditions i Used were
step 1
94C for 2 min
Cycle starts
94C 30sec
68C 5 sec (primer Tm 72)
72 5 min
35 cycles
Yeah I agree with Vairus..It depends on which kit or polymerase one is using.
R u doing a 2 steps PCR?
5secs for annealing is the time too short?
is it necessary to add another final extension?
I've done PCR without the extra final extension, but it probably is safer to include it. It takes only about 5 minutes, which on an entire PCR program to amplify 6 kb, which will take 4-5 hours, is not too much.
I also go for 15 seconds of denaturation instead of 94 (as your cycler climbs up to 94 and descends back, you also have denaturation). I believe this is better for my enzyme.
I also go for 15 seconds of denaturation instead of 94 (as your cycler climbs up to 94 and descends back, you also have denaturation). I believe this is better for my enzyme.
"15 seconds of denaturation instead of 94" ?
instead of 30...
I did PCR for approx 5KB fragement and used Expand Long PCR kit from Roche. There were 3 different buffers with differing MgCl2 concentrations and other characeterestics. I was not successful with Buffer 1 but Buffer 2 yielded a band. I used genomic DNA to amplify my sequence.
The PCR conditions i Used were
step 1
94C for 2 min
Cycle starts
94C 30sec
68C 5 sec (primer Tm 72)
72 5 min
35 cycles
Yeah I agree with Vairus..It depends on which kit or polymerase one is using.
R u doing a 2 steps PCR?
5secs for annealing is the time too short?
is it necessary to add another final extension?
sorry that was a typo .. I meant 45 seconds for annealing and not 5 seconds..apologies
that was a typo .. I meant 45 seconds for annealing not 5 seconds.
apologies