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