Change the annealling temperature. Increase the annealing temperature.
55 c annealing temp is low so it is very easy to get nonspecific bands.
How to get rid of non specific bands in pcr. G c too few amplification effect g c too easy to appear non specific bands. Non specific bands in my opinion can be reduced by altering temperature. The non specific bands could be from contamination of one of your stocks with foreign dna probably yours.
Atgc is best randomly distributed to avoid the sequence of 5 or more purines or. Reducing mg increasing annealing temp increasing decreasing extension time and temperature are different suggestions that can be made depending on where your un specific bands are. If for e g its 58 c use 68 c in the first cycle and then gradually bring annealing temp down to 58 c 1 c in each cycle and then rest of the 25 or 30 cycles on 58 c.
Increasing the blocking exposure time and or temperature at which you block using a higher the protein concentration in your buffer adding 0 1 tween to the buffer. Also with the receipt of your enzyme you can try using touchdown pcr. If the annealling temperature is too high you obviously won t get any priming at your desired sequence.
If you re having trouble with non specific binding consider. Perhaps most common pcr failure no bands at all on the ethidium. Or try to run a gradient for similar temp as well.
In the months to follow articles on the other types of failures will be presented. If this is a problem use new stocks always use autoclaved pcr vials and wear gloves and a lab coat. Try to change annealing or denaturation renaturation temp and its time.
In the range of tm values the selection of high annealing temperature can greatly reduce the non specific binding between the primer and the template and improve. Tips fpr pcr non specific banding rid off.