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PCR reaction ISSUES???? need help


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#1 massiri

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Posted 29 August 2011 - 09:58 PM

Guys I am stuck with PCR reaction for more than month could not figure it out neither my lab mates.

(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

#2 Ameya P

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Posted 29 August 2011 - 10:51 PM

Massiri,

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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#3 phage434

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Posted 30 August 2011 - 03:53 AM

Have you tried this pcr with a single 55 C annealing step? I've never gotten a pcr to work with less than a 48 C anneal, and 72 C seems awfully high. I think you are paying way too much attention to predicted annealing temperatures.

#4 massiri

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Posted 30 August 2011 - 09:49 AM

Thank you guys but i tried with 50 and 55 i got both same un needed products. I played with tm 1 several times but still got the same higher products. the problem is the expected product size is 104 but i am having higher. 117bp, 134bp and 162bp when i use 2 round of pcr with 2 cycle. when i threw the 3 oligo together and tm 50 i still got additional band around 71bp.
another problem i can not change the primer in this moment. out of time :(
Thank you guys

#5 massiri

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Posted 30 August 2011 - 12:45 PM

Guys I am stuck with PCR reaction for more than month could not figure it out neither my lab mates. (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


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Guys This is the oligoes sequencing




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