Dear Researcher,
I have used nested PCR to increase the specificity of Tetra Arms. Previously, I obtained results for another SNP using the same reaction conditions, and separate reactions (monoplex) verified that all primers are working efficiently. However, when I applied nested PCR, I observed no amplification; instead, the well glows. I have tried using a commercially available good quality ready mix and different PCR components, but the issue persists. I also tried changing the amplification temperature. Can anyone tell me the reason for this problem?
picture of gel is attached, well 1-6 = products from nested PCR, 7 = PCR product that was used for nestedPCR, well 8= ladder
Note: I am working on deletion polymorphism
I agree totally with Steve Baeyen about the dilution of the first round pcr. You must also be careful about the number of cycles of second round pcr. 10 cycles is 1000 times more product and 20 cycles is 1000000 times more product and eventually the pcr product anneals with other product molecules and you can get additive product much longer than the original. I woulf try diluted pcr product and only 20,23 or 26 cycles to see where to get clean amplification and run fewer cycles if the product is still smeary or concatenated
Steve Baeyen and Paul Rutland I quantified my first PCR product using a DNA ladder, estimating it to be 2 ng/µl. I then diluted it 10X and used 0.5 µl of this diluted DNA in a 20 µl reaction, resulting in a total of 0.1 ng of DNA in the reaction. I decreased the number of cycles to 25 for the second PCR, but despite these adjustments, the results remained the same (glow in the well). The product size is 306 bases. Could you suggest what might be going wrong?
using 0.1ng of a 306bp amplimer is to start the pcr with 3x10exp8 copies of your amplimer. 25 cycles could be an amplification of approx 3x10exp 15 molecules which is too much amplimer. Try starting with a 1/1000 dilution as
Steve Baeyen suggests and try running 15- 20 cycles of second round pcr
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude