Purified cDNA Clonning - (Jul/20/2008 )
I am going to clone a cDNA into pBluescript vector. I run 3 microlitre of the PCR product on 1% agarose gel to see the result ( pic attached ). Should I run all cDNA product on agarose gel and then extract my cDNA from gel before using it for clonning into my vector or use it the way it is ?
[attachment=5041:cDNA_PCR_Control_1.jpg]
I would try to optimize my PCR for a cleaner band before doing either of these. Even gel purification will leave you with many more copies of the short fragments in your purified DNA, and I would guess you will have difficulty cloning the fragment you want (which I presume is the long, weak fragment).
Can you use nested PCR?
Can you use nested PCR?
I'm afraid I can not use nested PCR but what do you propose to optimize my PCR ? I deluted my pcr product 10 times and then did the pcr thing using Tag polymerase to get more of the product but the band is the way you see.
Is there a software or method to use to have a sharper band ?
Have you tried different annealing temperatures? What primers are you using? This is almost always a problem with the primer specificity. Try raising the annealing temperature first. Make sure your extension time is sufficiently long. What is the template GC content?