PCR reaction ISSUES???? need help
Posted 29 August 2011 - 09:58 PM
(The reaction is about to make a template using three oligonucleotides for further use. The three olioges are: First oligo as a template 3'-5'. forward 5'-3'( Tm1 51, Tm2 70) and the Reverse 5'-3' ( Tm1 44, Tm2 79). I did 2cycle of PCR using 42 as Tm1 and 72 as Tm2 and Green Go Taq. However I got lower bands that i dont need ( the meaning, secondry products that i dont not need and it is lower than my product. So I gave it another shot using 2 round of pcr and this is what i did:- I use the 3-5 olig as a template and i just used the reverse, In this reaction i got the product of interest and the expected fragment, so i used it as atemplate for the second round and i used 48 as Tm1 and 72 as Tm2 ( 2 cycles) but i got secondary products that are upper level of my fragment of interest even if they are so thin but still. I am just really confused and cannot do this reaction any more. PS I did PCR purification, and I used denaturing page 8% Poly Acrylamide gel to analyze my products)
Thanks in advance
Posted 29 August 2011 - 10:51 PM
Looks like the second primer you are using is not very specific. Try and add two to three bases to the primer and it should work out. If you cannot change primers for any other reason, increase your Tm1 from 48 to 50 or 52oC. That should reduce the non specific bands to a certain extent. Hope this helps.
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Posted 30 August 2011 - 03:53 AM
Posted 30 August 2011 - 09:49 AM
another problem i can not change the primer in this moment. out of time
Thank you guys
Posted 30 August 2011 - 12:45 PM
Guys This is the oligoes sequencing