Long-range PCR

[yozyo123]

Hi, I've been trying to PCR out a 8kb fragment from a huge 11kb plasmid using Clontech's Advantage 2 polymerase mix. the 1st 2 times it works..but subsequently it stopped working. I've been getting smears on my gel & eventually no bands with repeated PCR attempts. I've tried to restart on completely new tube of primers,dNTPs, enzymes & buffers...& even a different source of template stock. Still no 8kb bands. My positive control works ok though. I can only say probably the 1st 2 times when i got it, it's a fluke. Can anyone help me out here? Thanks.

[niki_nje]

you didn't change any conditions whatsoever?

[admin]

Hello,
there are some great papers on long range PCR that I would look into. I would also double check your water, buffers, and pipettes (use PCR specific ones) as you could easily have DNase or other junk messing around with your experiments.

Also, try to check your DNA template on a gel again to see if its ok (no smearing etc).
One final thing, try a some new primer dilutions fresh from the freezer instead of your old one.

Please also post a gel picture as we can help more that way if we see whats going on :0
welcome to the forums
cheers

[moleculardude]

How is the long PCR going?

[yozyo123]

Thanks for your help people... I've managed to get my pcr working again. The reason was pretty silly,to me at least. All those times when it didn't work, I've tried to restart with new water,wiped my pipettes...but they didn't work. I did a positive control (~2kb) & it works to give a band...so the problem's not the machine. those smears I initially have are probably degradation. Then i don't get anymore bands for subsequent pcr attempts...i think the enzyme's lost it's robustness, being only good wenough to pcr smaller fragments(like my 2kb positive control...i don't have a bigger control) & not the big ones(like my 8kb). then i used up my pcr enzyme. with the new kit, i got my PCR to work again. So it's the enzyme problem, i concluded.

[kwak050582]

I am having a similar problem where I am getting low molecular weight smears for my PCR. I am trying to amplify a 3kb segment. Primer 3 was used to calculate the Tm adn design the primers. Left primer was about 62 and right primer was about 61. I am consdiering increasing annealing temperature (last set of annealing temperature used were 2 degrees below and 2 degree above the stated Tm). Also I 've added betaene (from sigma) which is suppose to help amplify regions of high GC content and will consider decreasing Mg concentration or decreasing annealing time to get rid of some of the small product sizes. Are there any other suggestions? Thanks

[aneekmvk]

Recently I am trying to standardize a long range PCR (10-12Kb) using the Expand Long Template PCR System kit from Roche, Germany. Its a long range for detection of an inversion resulting from a homologous intrachromatid or intrachromosomal recombination between a 9.5 kb region in intron 22 (int22) of F8C gene (int22h1) and one of two almost identical copies (int22h2 and int22h3) in the same gene in Hemophilia A patients. I have intended two kits and the fact is one kit is already finished while standardizing and no positive result has come. After lots of variations I am only seeing the smears in the 0.6% to 0.8% Agarose Gel. No band has come yet. I am following some papers regarding this and using there protocols (Polakova et al. 2003; Liu et al. 1998). I am confused now. I dont know after following some already standardized protocols also why the bands are not coming? Can anybody help and give me suggestions please? Also I want to know how much PCR product I should run in the Gel to get rid of the smears? Should I dilute the products with water and then load it?

[davidflora]

[Only registered users see links. ]Long range PCR typically refers to the amplification of DNA fragments in excess of 5 kb, using a blend of thermostable DNA polymerases that allows for the amplification of longer fragments than those that can be achieved with a single enzyme. Enzyme blends consist primarily of an A-family DNA polymerase (e.g. Taq), combined with a small amount of a B-family or proofreading DNA polymerase. Both enzymes possess 5’ – 3’ DNA-dependent DNA polymerase activity, but only the B-family polymerase possesses 3’ – 5’ exonuclease or proofreading activity.