I am needing help cloning a sequence into a vector.
I have this 1.5 Kb sequence to which I want to add 3 bases before the start codon.
I had a primer forward to amplify this sequence.
what I did to add these 3 bases was just adding 3 bases in the primer sequence like shown below:
Old primer was: 5' ctt a'gg ctt atg gcg aac ctt ggc tac - 3'
New primer: 5' ctt a'ag ctt (atc) atg gcg aac ctt ggc tac 3'
The difference between the two primers is the atc added just before atg.
After sequencing the plasmid extrated from postive colonies of top 10 cells transformed with pgem vector to which the pcr insert was ligated, I didn't have the sequence with the atc, but the sequence was as if I didn't change anything.
Now I don't have a clue of what happened, I wonder if there was a loop in the primer, as the rest could anneal but not the atc.
May be a contamination, but I don't think so. would it be better to remove the ctt just before the atg in the original primer and replace it with atc instead of addind the atc?
or in your experience what do you thing was wrong?
What would you do if it were you doing this??
please read and help.
Edited by Ewan McGregor, 23 March 2010 - 02:14 PM.