PCR products stick in gel wells, PCR problem
Hi Guys:
I performed PCR to confirm if my gene knock-in is working fine. The templates are yeast genomic DNA. Primers' Tm are 45C and 60C, and I set annealing temperature at 55C for 30 second. The target PCR products should have 2400 bp. The genomic DNA has been 100-fold diluted before PCR, which is about 1-5 ng per 20ul reaction.
I have used new 1x TAE running buffer and another PCR product which the size is known (3300bp) as control.
But when I run a gel, all my PCR products just stick in wells and they almost didn't migrate. My control PCR product (3300bp) migrated at the right size. Marker looks normal.
Please give me any suggestion.
Do I need to dilute more for the template genomic DNA?
Thank you for the help. I really appreciate.
Jun-
I performed PCR to confirm if my gene knock-in is working fine. The templates are yeast genomic DNA. Primers' Tm are 45C and 60C, and I set annealing temperature at 55C for 30 second. The target PCR products should have 2400 bp. The genomic DNA has been 100-fold diluted before PCR, which is about 1-5 ng per 20ul reaction.
I have used new 1x TAE running buffer and another PCR product which the size is known (3300bp) as control.
But when I run a gel, all my PCR products just stick in wells and they almost didn't migrate. My control PCR product (3300bp) migrated at the right size. Marker looks normal.
Please give me any suggestion.
Do I need to dilute more for the template genomic DNA?
Thank you for the help. I really appreciate.
Jun-
Popular Answers
1) the gell loading buffer which you used for loading the sample is not proper.
if you have used he same gell loading buffer for the markers and your control then this possibilty is ruled out
2) PCR is not being carried properly.
this mght be because the designed primer pairs are not proper. Tm difference should not be more than 5 degree celcius. In your case the Tm of your primer is 45 degree and 60 degree but you have set the anneling temperature at 55 degree. In such case only the primer with the Tm 60 will anneal and not the primer of 45 degree. this will result in amplification of single strand.
But as in your case you are getting a bright band, this could be due to genomic DNA; indication negetive PCR amplification.
So i will suggest that check the gell loading buffer and redesign your primers with Tm difference not more 5 degree celcius.
If you want to check if your PCR product is single strand amplification then perform PCR in two sets. in one set add both the primer and in the another set add only the primer with Tm 60 degree. keep the condition same as you mentioned above. keep annealing temperature 55 degree for both the set. observe the result.
Maybe anyone else from here has another idea?