negative control well 300 bp band - (Aug/16/2013 )
Hi there ,
I have a question regarding to primer dimer in control sample .
I want to conduct an expression analysis experiment of NK-lysin in gibuna crucian carp fish using TAKARA (Q-PCR) instrument and syber green dye.
I tried to design primer either with using software like primer 3 , primer express or manually , but analysis of results by beacon designer free edition showed a primer dimer and 2 nd structure formation. even though I decided to conduct Q-PCR with the best candidates (Denaturation : 95 C for 10 sec. ; Annealing : 60 C for 5 Sec. Extension : 72 C 1 min.) and I found amplification curve in negative control wells.
I tried to make gel electrophoresis to the product of Q-PCR but surprisingly I found bands of 300 bp (negative control wells) , I suspected genomic DNA contamination so I discarded all of my (primer , water , and Syber green as well ) and used new ones but still my problem is the same high molecular weight bands.
My questions are :
1- How could I design primers without dimers or 2nd structure
2- Is it possible to increase the annealing temp. to avoid these 2nd structure or primer dimer
3- what is the maximum annealing temp I can try to avoid this dimer formation
Attached jpeg file show the high molecular band in control well (no template well)
Please any comments , advices or answers are welcome
Yours
Haitham
Hi Haitham,
From your gel picture I am not sure, what is the size of PCR product that you are expecting. But yes, you can definitely increase your annealing temp to 65 degrees and even above, if you are getting dimers, as long as your product of interest is also seen.
Ameya P on Sat Aug 17 11:14:26 2013 said:
Hi Haitham,
From your gel picture I am not sure, what is the size of PCR product that you are expecting. But yes, you can definitely increase your annealing temp to 65 degrees and even above, if you are getting dimers, as long as your product of interest is also seen.
Thanks dear Ameya P
Actually those wells in attached electrphoresis JPEG file are only for negative control (No cDNA) samples but I still find bands of long sizes although I changed all PCR components , So I am asking is that possible to find these (300 - 600 bp) bands in negative control wells ??.
Is it possible to find a primer dimer of > 400 bp ?
What exactly is your negative control, no cDNA.... 1) RNA sample that has not undergone RT, or 2) no template added to your PCR mix.
If 1) you have gDNA contamination on your RNA, you'll need to treat with DNase, also you should have intron spanning primers to avoid gDNA amplificaiton.
If 2) you have PCR contamination somewhere. Primer dimers will not be that MW (unless your primers are >200-400bp)