cloning the promoter from genomic dna - (Feb/25/2006 )
i am trying to clone the promoter (around 1 and 1.5kb) from genomic DNA (mouse cell line) and then to clone it in the pgl4 vector (Promega) for lucifease assays.
First i optimized the conditions with TAq polymerase, and then switch to PFU from Promega. and this is where the problems started because the maximum size of the PCR product that i can get is 700bp with Pfu.
i tried almost everything, but i can not get more then 700 bp.
is there any better Pfu polymearase?
like turbo pfu from stratagene?
any ideas or suggestions are more then welcome....
I cloned my promoter with exTaq from Takara... worked well... Cloned 2.5 kb out of a bac clone...
Hope that helps
thanks, i will take a look at takara. i am just afraid of mutations bwith such a long fragments...
BTW, what is the difference between using bac clone and genomic dna as a template for the PCR?
is bac less complex?
Exactly, the bac is less complex... Helps to avoid mispriming...
the ex taq is the polymerase from takara that is supposed to give long, relatively error free amplimers... my sequences compared well to what was found in the genome-sequencing project... (very few "mutations")
Stratagene has indeed better variants of Pfu, like PfuTurbo and PfuUltra. Apart from this, you can also search around the web for other companies that sell high fidelity enzyme mixes (taq + proofreading enzyme) or pure proofreading enzymes. There's heaps of them (phusion, pfu, pfx, pwo, vent, ...). But 1 to 1,5 kb isn't too big, have you tried magnesium titration?
Btw: with pure proofreading enzymes, the chances of having sequence changes in your clones is smaller, but it's still existing. I cloned a 2,2 kb gene once and only got two clones of which one had an error, pcr enzyme used was 'expand high fidelity' from roche (which is a mix of taq and a proofreading enzyme).
Pfu is able to produce fragments longer than 700 bp. Do you mean that during your experiment using Pfu you're getting a 700 bp product?
If that is the case, the problem likely lies with your conditions, PCR program, or primer(s), and not the enzyme used.
Thanks guys! I am planning to order either Pfu turbo (Stratagene) or Pwo super yield from Roche.
THIS PWO IS A NEW PRODUCT FROM ROCHE. anybody tested it?
I also tried high fidelity pcr system from roche and it worked, also with the biggest fragment (1.5kb).
i have impression is more PFU the problem (believe me i tried almoste everything, all the conditions, but nothing...)
i think the problem is that pfu *at least that one that i used (promega) is very slow, and it seems that the one from stratagene needs fewer cycles and less elongation time.
at the moment i am doing the ligations and will see in few days if I get any right colonies...
keep u update!
thanks once again!
Yes, pfu has a slower elongation rate than other polymerases -- I recall reading you should allow two minutes per kb.
See, for example here:
Usually the extending step is performed at 70-75°C. Pfu DNA Polymerase exhibits lower extension rate than Taq DNA Polymerase, so 2min extention time is recommended for every 1 kb to be amplified.
so after one month of cloning, sequencing, cloning.... I have realized that using 2 diferent polymerase, Taq that was a part of ROCHE HIGH FIDELITY KIT and PFU from Promega, i got several clones with mutations always at the same place!!!!!!!!
So it can noty be the polymerase. Of course, I got some mutations specific only for ROCHE kit, but some mutations were presentalways at the same site in different clones, using 2 different polymerase.
So, I think it is the genomic DNA that is mutated (is it polymorphisam?).
And what do you do when you realize that your cloned promoter has a mutation due to the template (in my case is the genomic dna).
do u proceed with luciferase assay and hope that this site where is the mutation is not important???
is there any other way to avoid this problem?
maybe by using BAC clone as a template, instead of the genomic dna?????
try using Invitrogen Pfx Kit
i amplified my segments
has higher proof reading and better quality PCR