Poor PCR result - (Aug/01/2001 )
Hi every body,
i am woking on cloning and charecterization of a mycobacterium gene.initially i got PCR product butligation was unsuccessful.now i want more PCR product for ligation but problem is this that now i am gettingvery poor amplification with nonspecificity also. Yield is so low thatis why i am not able to ellute my pcr product for digestion as well as ligationi am very confuged because other PCR are coming so reagents can not be doubtfull.i isolated mycobacterium DNA from simply boiling prep andother lenthy procedure also.i also made fresh dilution of Primer from stock.please suggest me wheather problem is in DNA or some where.if you have good protocol for mycobacterium genomic DNA isolation ,please write me.
It you have a very weak band, but are quite sure that it is the right one,you can cut out the band, put in an eppendorftube, add 10 microliter water, crush the gel in the tube, leave the tube for a while (over night 4 degrees),spin down and take 5 microliters (or more or less) of the water now on top of the gel in the tube. This water will now contain enough DNA for you to run a new PCR with this DNA as you template!
Change oligo or increase annealing temperature. Ligated in TAclone and identify it. Your issue is simple and common.