PCR常见问题的精辟总结.docVIP

PCR常见问题的精辟总结--耶鲁大学 作者：佚名 来源：耶鲁大学 时间：2007-9-5 Troubleshooting for PCR and multiplex PCR Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles. Component volume final concentration 1.autoclaved ultra-filtered water (pH 7.0)20.7micro;L- 2.10x PCR Buffer*2.5micro;L1x 3.dNTPs mix (25 mM each nucleotide)0.2micro;L200 micro;M (each nucleotide) 4.primer mix (25 pmoles/micro;L each primer)0.4micro;L0.4 micro;M (each primer) 5.Taq DNA polymerase (native enzyme)0.2micro;L1 Unit/25 micro;L 6.genomic DNA template (100 ng/micro;L)1.0micro;L100 ng/25 micro;L * The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2). 问题解决： 1. I get (many) longer unspecific products. What can I do? Decrease annealing time Increase annealing temperature Decrease extension time Decrease extension temperature to 62-68ordm; C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM. Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s) Combine some/all of the above 2. I get (many) shorter unspecific products. What can I do? Increase annealing temperature Increase annealing time Increase extension time Increase extension temperature to 74-78ordm; C Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Take less primer Take less DNA