problem in sequencing (View forum version)


Posted 02 March 2009 - 07:10 PM

Hi all,

I gave the clone for sequencing (M13 primers given), the forward primer gave correct sequence but the reverse primer gave non specific amplication.
then I tried to sequence it with the reverse gene specific primer, then i got the correct sequence but the last 70 bases were not amplified.
what could be the problem. but the complete gene size of 900bp was observed during the PCR amplification and succesive expt

appreciate your suggestions


Posted 02 March 2009 - 07:50 PM

Your vector may not have the correct M13 reverse primer site. There are two forward and two reverse sites commonly used, both named M13, and not all commonly used cloning vectors have both. The gene specific primer would bind internal to your sequence, and this would likely explain why you do not see the last 70 bp. I'd guess your clone is correct. What is the vector you used? Can you check to see that the primer site is really present for the primer you used in the reverse direction?


Posted 03 March 2009 - 05:46 AM

Hi phage
The vector I used is PTZ5/7 RT provided with the insta TA cloning kit,It definately has the M13 site.

thanx for the reply


Posted 03 March 2009 - 06:31 AM

just a guess, but was your unspecific sequence e. coli or vector derived??? Maybe you have co-amplified some DNA form the host cells and the M13 reverse primer had a higher affinity to the ecoli seq.....or you have a mixed culture of a sucessfully transformed plasmid and a self ligation plasmid so the M13r gives you the vector seq.....

And the missing 70bp: do you mean at the reverse-primer end of the seq?? Because when seq the first 100bp after the sequencing primer are missing. This is one of the reasons why you use seq primers located at the vektor to get the full seq of your DNA fragment of interest....


Posted 03 March 2009 - 06:44 PM

I guess you can design another forward primer in your insert region, maybe in the middle, do the sequencing agian to know what happens.