I have cloned a 2.7 kb insert into the pCAMBIA1300 vector and confirmed the cloning by restriction digestion and plasmid PCR. However, after Sanger sequencing with M13F and M13R primers, I am obtaining the same sequence in the first 1000 bp at the 5' end, and both sequences appear on the negative strand. I can't understand the problem.
One possibility is that the m13 primers are , in fct, both the same primer due to a labellling or pipetting error. You could try running a pcr with the m13 primers to check that they are paired and not the same
@Paul Rutland Thank you
I already checked it by pcr and got the desired band with m13 F/R primers.
Abhilasha Singh
are you purifying your pcr product with exo-sap before sequencing please?
Are you sure that you don't have 2 inserts in your clone that are inverse?
@Michael J. Benedik Thank you
Yes I am sure. Confirmed by restriction digestion.
Abhilasha Singh
Please consider this theory,
Exo sap only works on single stranded dna so it is very useful in removing pcr primers from a mixture with double stranded dna (product and template)
Many years ago I worked on an exception to the usefulness of exo1.......so if one of the primers has the ability to form either inter oligo G quadruplex or intra oligo G quadruplex then after the last cycle of pcr the primers now have some DS dna present stopping the action of the exo.
What does this mean for your sequencing?
It means that if your forward primer has sequence like GGnnGG then the GGs form a loop and after exo digestion then most of primer F still exists in your pcr mix while the R primer is destroyed.
In sequencing the Ta is 55c so some quite short leftover primer can still work as a sequencing primer. So when you add sequencing primer to your amplimer the F primer and remainder F primer will work. The reverse sequencing does still have a lot of F primer left and this may take over the sequencing reaction so you get 2 forward sequences.
A simple test for this situation is to exo sap the pcr product and then run the sequencing reaction WITHOUT any added primer. If you get a good sequence then primer is remaining in the pcr template and is probably just swampling the reverse sequencing reaction.
Check your primers for GG, GGC, or any other G quadruplex forming sequence near the 5' end of the primer. Other loop forming sequences like cccnnggg may also give the same effect.
best wishes
Paul
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