sequencing trouble - (Nov/15/2011 )
i'm trying to figure out the problem with sequencing my insert..i have cloned a 3kb fragment into a vector of around the same size...inorder to confirm i tried a pcr by amplifying 5 fragments from this 3kb fragment....n i got the products of expected sizes..but when i try to sequence the whole plasmid using vector specific primers that should sequence only my insert..instead i get the vector sequence only...what could this problem be due to??
lashed on Tue Nov 15 20:19:43 2011 said:
i'm trying to figure out the problem with sequencing my insert..i have cloned a 3kb fragment into a vector of around the same size...inorder to confirm i tried a pcr by amplifying 5 fragments from this 3kb fragment....n i got the products of expected sizes..but when i try to sequence the whole plasmid using vector specific primers that should sequence only my insert..instead i get the vector sequence only...what could this problem be due to??
Did you put it on gel?
(put the vector with the insert on gel).
It does seem weird what you tell, but many things can go wrong, contaminated PCR or maybe you used the wrong primers or...)
How did you isolate DNA from your transformed cells? Carryover DNA from the transformation can be amplified by PCR, and make it look as if there is an insert. You can distinguish this by PCR using a primer to the vector and a second primer on the insert.
thanks for the reply... well iv already tried both the methods...problem is my insert n my vector are alost of the same size..(the reason why i tried amplifying a known fragment from the insert....
are you sure about the orientation of your primers?