I'm trying to amplify a 8kb fragment (gene+promoter) form genomic DNA, using roche Expand™ Long Template PCR System. My primers are ~21bp long and the annealing temperature is 2 degree centigrade below Tm. The DNA template is placenta DNA and I use 50ng in each 50ul reaction system. The rest are all according to the protocol. But I can not get band at all.
Who have ever used roche Expand™ Long Template PCR System to PCR a large fragment from genomic DNA? Can you share with me some experience and tip? Thank you so much!
Please begin 5 degrees below the Tm as starter. Which buffer you are using, with or without detergent? Is your template GC-rich?
Dear Syed A Ali,
I tried 5C below Tm but still didn't work. The CG content is ~44% for my primer. The buffer I use in the kit has 0.5% TWEEN 20.
Dear Artur Burzynski,
The Tm for my primers are 59 and 57. I design a new pair, whose Tm is 63, but haven't tried yet. I'm sure the aimed sequence is 8kb. I get it from NCBI gene bank.
I just bought the Roche kit, if it still doesn't work, I will try the one you mentioned.
Roche has given preparing master mix and cycling conditions in its template. Use that only. It will work. No need to change the annealing temperature.
Just to make sure: Are you allowing at least 8-9 minutes for amplification? Did you try a temperature gradient to make sure that the calculated amplification/melting temp is accurate? I was able to amplify 11 kb with a Takara enzyme, but it took me a few trials.
http://www.sciencedirect.com/science/article/pii/S1096719211000035
Dear Sudhanshu Pandey,
Yes I follow the protocol and didn't add anything except the reagents in the kit.
Dear Artur Burzynski,
I got my sequence in the following way:
Open pubmed, choose gene and type my gene name. For human, there is only one under the name I search. Then I chose FASTA to get the whole sequence I want. I think it is genomic DNA. Hope I am right.
Dear Isabelle R Miousse,
I use 8 min for elongation. As the Roche protocol says, 8 min can cover up to 10kb. I didn't try temperature gradient and just used 3 or 5 degree below my Tm. Is it a must to try temperature gradient?
Thank you for your enzyme recommendation. Since I just bought a new Roche kit, I have to use it for a while. If still can not work, I will try Takara.
Definitely try different annealing temperatures (i.e. a gradient will be the most efficient). My issue was different than yours, but when trying to amplify targets from mouse fecal DNA, I had to go way below the Tm of the primers for the reaction to amplify my product.
Shuaichen, I think you might already finished your work. Here I use your post to write down some of my experience for the other readers.
1) The most important thing for the ROCHE kit is that EDTA in the template should be removed;
2) Buffer 3 is most powerful;
3) A nest PCR might work.
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