problem in sequencing
Posted 02 March 2009 - 07:10 PM
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
Posted 03 March 2009 - 05:46 AM
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
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