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Condition for amplifying >6k PCR - Long PCR amplification (Oct/11/2005 )

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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!

-axisy-

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.

-vairus-

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.

-Watson-

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

-axisy-

QUOTE (Watson @ Oct 11 2005, 05:24 AM)
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?

-axisy-

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.

-vairus-

QUOTE (vairus @ Oct 12 2005, 01:10 AM)
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.


"15 seconds of denaturation instead of 94" ?

-axisy-

instead of 30...

-vairus-

QUOTE (axisy @ Oct 11 2005, 11:19 PM)
QUOTE (Watson @ Oct 11 2005, 05:24 AM)

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

-Watson-

that was a typo .. I meant 45 seconds for annealing not 5 seconds.
apologies

-Watson-

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