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Need help with PCR reaction as soon as possible?
Guys, I am stuck with PCR reaction for more than month. 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, secondary 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 a template 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% Polyacrylamide gel to analyze my products.
Re: Need help with PCR reaction as soon as possible?
Could be Primer Dimers in the first round of PCR--desalting through a spin column (sephadex or acrylamide) gets rid of them, as well as diluting them out.
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