what was the main problems you face? dose the concentration of the genomic DNA affects the results? any advises or recomended reading about the procedure.
2007-09-01 10:47:28 · 2 answers · asked by dan 1 in Science & Mathematics ➔ Biology
That isn't too, too long. At this point your biggest concern is going to be using a good, hi-fidelity polymerase. Ultra PFU is my favorite for these applications (Taq and its derivatives get a little messy after 10kb or so). You might also want to do more cycles during the PCR, and remember to allow for a long enough elongation time for such a large fragment. Genomic DNA shouldn't affect your results here anymore than during a smaller PCR, so I wouldn't be too concerned about that. You should try to make sure it is as clean as you can possibly get it, though. Alcohols and phenol can be a big problem.
A very long gel will also be helpful in getting good resolution at that length. I am pretty lazy, and so I would also probably drop down to a .6% gel. Get a good, reliable high molecular weight DNA marker too. Pulsed field gel electrophoresis is sometimes employed for larger fragments too, but mostly for DNA well over 100kb in length...I don't think you need to consider it here. Good luck!
2007-09-01 11:15:36 · answer #1 · answered by BLLYRCKS 5 · 1⤊ 0⤋
Billy's advice is good. If you want to spend for a kit, Roche (formerly Boehringer-Mannheim) has a couple of high-fidelity long product kits available. I believe they use as an enzyme a combination of Taq and Pfu to get Taq's speed and Pfu's proofreading. Just make sure you sequence your product both ways to check for errors when you're done. It's not all that much more troublesome than normal PCR.
2007-09-01 19:06:31 · answer #2 · answered by John R 7 · 0⤊ 0⤋