working with high GC content template
Posted 10 July 2003 - 03:27 AM
Total number of bases is 1341 with % C+G = 64.73 . I designed a pair of primers with Xba1 and Sac1 site respectively, they are 30 & 31nt respectively, both 22 nt complimentary to the target sequence. GC% are 46.7% & 38.7%.
When I doing PCR, only about 1/3 buffer concertration give the righ product, with the buffer concertration increase, the product is lost! Why?
Another problem obsessing me is I fall when I enlarged the system to 100 ul with 1/3 buffer, while I can get PCR product with a 15 ul system.
I think the high GC content is the problem, if I redesign a pair of primer, what should I take care of? More over, because I am PCR a intact gene, I have to cope with the ORF.
Posted 14 July 2003 - 03:05 AM
I have no clue about your problem with the product loss when using the 1 x concentration of your buffer, in my opinion reaction conditions in molecular
biology are more intuitive than rational.
E.g. I just play with the annealing temp and the MgCl2-concentration until
I get the desired product.
Anyway, the problem in the scale up of the PCR reaction seems to be the
programm of the thermocycler. You've expanded your reaction volume by
a factor of seven, so the temperature profile in your large PCR-reaction will
not be the same as in the small reaction, so you also have to increase the
length of the denaturation-, annealing- and synthesis-cycles a little bit.
Posted 15 July 2003 - 01:33 AM
Also, I have observed that when you increase the reaction volume, the amount of the desired product does not increase proportionally using the same PCR program....in general I obtain less product.