I tried to preform Gibson reaction and insert 3 fragments into a backbone. I used the neb builder to build primers for the fragments. got clean sharp bends. cleaned the backbone from restriction and gel. Got alot of colonies, so i planed different primers for each of the fragments. Took only the ones that were positive for all 3 fragments but when I sequence them it seems like I only get 2 out of 3. while when I use the pcr with the specific fragment primers I get all 3.
how is that possible? what should I do?
Are you using for sequencing the same PCR primers that confirmed your positives in the gel? Do your bands have the absolutely correct size for the fragments you've amplified?
I am using the same PCR primers for sequencing. I get the correct size bands but they seem to be very weak when I run them from PCR, even after 35 cycles.
Dear Lior,
Depending on the insert position in the final construct and size of the respective insert, you should try a PCR with a combination of a backbone- and insert-specific primer. This could confirm if your insert was actually assembled into the vector. A weak PCR signal could originate from your original, unligated PCR product (of the insert) which was present in the Gibson assembly reaction mixture. The fragment was most likely cotransformed with the plasmid and not completely degraded at the timepoint of the PCR.
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