【在 B*******d 的大作中提到】 : Please do nested PCR to increase PCR yield. : Examine the promoters first. : You can design your own primers using the same principle from meth primer. : : 区?
How do you claim a reduced expression level of your candidate genes in your mutant? by qPCR or another approach? what's the control? What is the size between two flanking markers?
there are about 10 gene between two flanking marker. I did the RT-PCR and qPCR to check the expression of candidate gene bewteen the mutant and wild type.
your
【在 C*******h 的大作中提到】 : How do you claim a reduced expression level of your candidate genes in your : mutant? by qPCR or another approach? what's the control? : What is the size between two flanking markers?
1) RT-PCR not quantitative 2) for q-PCR, you would need to have several proper controls at least two genes as your reference 3) I would suggest you sequece the whole region since you have approximately 30 kb. 4) For certain region with low GC content, you can amplify 5-6 kb fragments (it is should be easy in most cases) to overcome the PCR obstacles and then design primers for sequencing. Good luck