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Long PCR and genomic DNA isolation problems - (Nov/16/2009 )

Hi, i'm trying to PCR a big fragment, approximately 28kb, out using mouse genomic DNA. I failed my pcr and would like to ask several questions pertaining to it.

1) I isolated mouse genomic DNA using Qiagen blood mini kit. The kit says that it can purify genomic dna up to 50kb, but predominantly its 20-30kb fragment. I did run a gel for the genomic dna isolated and they show a strong band around 20-30kb, with a little of smear above and below the band. I thought that as long as there is some genomic DNA larger than 28kb, it shouldn't affect my long pcr right? But just in case, i was thinking to isolate larger genomic DNA without buying another kit. I saw a standard protocol at this link, http://www.genome.ou.edu/protocol_book/pro...tIII.html#III.H. Does anyone know if this protocol allows high molecular weight genomic DNA isolation? Or does anyone with prior experience of cloning such large fragment knows if the genomic dna cloned via the qiagen blood kit is good enough?


2) Regarding the long PCR, i'm using Epicentre master amp extra long pcr kit. The cycling conditions are as follows: initial 94deg 1 min denaturation, 15 cycles 94deg 10 sec denaturation, 62deg 1mins annealing, 68deg 28mins extension, 15 cycles of the same thing but with extension temp increased by 10sec every cycle.

The annealing temperature of the primers are 66deg. I'm using 150ng genomic DNA (they recommend 100-500ng for 50ul reaction), 0.2uM primer each (final conc) for a total of 25ul reaction.

So my first attempt failed. I suspected its because of my primers, which was supposed to be 25bp and 24bp long (blasted and checked for dimers and other secondard structures). I ordered the primers with additonal 12bp at each end to include restriction site and GC over hang, thinking that it should be all right since we do that often for typical PCR.
Does anyone know if that could be the problem? Anyhow, i juz ordered the primers again, this time without any additonal sequence. I'm intending to retry the pcr again but i would like see if anyone find any problem with my pcr conditions or cycling parameters, or have any advice on it.

I'm thinking for the next pcr to perform a step down, to start with annealing temp of 70deg, decrease 2 deg every 2 cycles till 60degs. That is in addition to the cycling parameters i'm already using. Also, i'm going to include a final 10mins extention at 68deg.


Any other suggestions will be much appreciated. Sorry for the long question. Thanks

-krystle-

hi,
for what do you need such a big fragment? It is difficult to get such big amplifications, expecially if starting from a big genome and if you need it mutation-free. regarding your polymerase, they claim to get up to 40kb, but it's lambda... A colleague of mine also got >30kb, but was from a BAC. I used the "expand long template" system from roche, but I never got such big fragments, and moreover had to get back when I saw that it introduced a number of mutations (I had a reference sequence...). I then proceeded with performing shorter PCRs overlapping unique restriction sites that I used for subcloning and then ligate to the other parts of my big piece.

If you need to go on with your procedure, I would advice you to read carefully the troubleshooting part of your polymerase, that should advice you to add some DMSO or similar (such big fragments are for sure involved in some secondary structure), increase cycle number, add Mg++ etc.
Moreover, since in your first cycles not the whole primer will anneal, you have to lower the Tm (-restriction site).

Last, to isolate (relatively) High Molecular Weight DNA I used a tip of the tail of the mouse (less than 3mm), combined with 100ul of:
10 mM Tris-Hcl, pH 9,0
50 mM KCL
0,5% NP-40
0,5% Tween 20
0,1 mg/ml proteinase K
and let shake (gently) o/n @ 55C, followed by PK inactivation @90C 10mins. then cetrifugation to remove debris, Phe/Chl extraction, isoprop/sodium acetate precipitation, EtOH wash and resuspension in TE (takes long, better @50C with gentle shacking). Never vortex, just invert

Hope this helps

-piemmea-

since your genomic dna is fragmented, are you sure that your priming sites are present in the fragment?

-mdfenko-