my gene of interest size is about 4kb. I used hi fidelity taq pol as well as Q5 from NEB but still its happening. my forward primer composed of a 4 adenosine then restriction site followed by 6x his tag then enterokinase site and forward primer of amplicon.(63 bases) and reverse is normal,4 adenosine then restriction site followed by reverse primer of amplicon( 30bases). i changed all the reagents to improve pcr but same result follows. what should I do now?
Hello, may be it is an electrophoresis issue. Are you doing a 1% agarose gel? your ladder seems to have a band" also.
i would make an 1,5% agarose gel, then i would run an electrophoresis at 80V for 40-50 minutes and finally i would make a post-cast staining with etidium bromide for 15 minutes.
Hi Gourab,
Firstly, your gel seems to be overloaded.
Secondly,I think, it is mostly an electrophoresis issue and thirdly your PCR purification product doesn't look clean! It still has a huge amount of unreacted primers.
Load less DNA, use fresh TAE and also clean the tank (I know how dirty they used to be!!) Did your cloning work?
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