Sequencing result is vector self-ligation while bacterial PCR identification was - (Mar/26/2008 )
Hi everybody, I have done TA cloning these days. I found some sequencing results are vector self-ligation while bacterial PCR identification was correct. The primers used for Identification blong to insert DNA itself. I am so anxious to got some advise from you.
Were you careful in picking individual colonies?
Perhaps you accidentally picked more than once colony. So when you do PCR with a gene-specific primer, it amplifies correctly. However, for sequencing you are using a primer within the vector so everything will sequence. Is your sequence messy to indicate a mixed product?
Perhaps you can just sequence your PCR product. Only problem is you will lose 50-75bp of sequence on the end where you amplified with your gene specific primer.
If you can redo the PCR, use both forward and reverse primers within the vector. Run it out on a gel and make sure the size is right for your insert, then do PCR cleanup and sequence that.
oh, one more thing.... did you have a negative control for your PCR? Perhaps a reagent is contaminated and the band you want is actually just contamination across all the samples. As long as you had a few lanes not show the product, then that is basically your negative control.
Perhaps you accidentally picked more than once colony. So when you do PCR with a gene-specific primer, it amplifies correctly. However, for sequencing you are using a primer within the vector so everything will sequence. Is your sequence messy to indicate a mixed product?
Perhaps you can just sequence your PCR product. Only problem is you will lose 50-75bp of sequence on the end where you amplified with your gene specific primer.
If you can redo the PCR, use both forward and reverse primers within the vector. Run it out on a gel and make sure the size is right for your insert, then do PCR cleanup and sequence that.
Maybe you misunderstand my means. primer for sequencing is M13, while primers for bacterial PCR identification is insert DNA itself. Thanks!
Perhaps you accidentally picked more than once colony. So when you do PCR with a gene-specific primer, it amplifies correctly. However, for sequencing you are using a primer within the vector so everything will sequence. Is your sequence messy to indicate a mixed product?
Perhaps you can just sequence your PCR product. Only problem is you will lose 50-75bp of sequence on the end where you amplified with your gene specific primer.
If you can redo the PCR, use both forward and reverse primers within the vector. Run it out on a gel and make sure the size is right for your insert, then do PCR cleanup and sequence that.
Maybe you misunderstand my means. primer for sequencing is M13, while primers for bacterial PCR identification is insert DNA itself. Thanks!
Exactly...because you are using primers that are complementary to your insert, you may amplify what tiny amount of a 2nd colony you picked by accident.
Imagine you have two colonies growing right up against each other so they look a lot like 1 big colony. One colony is an empty vector colony and the other is a colony containing the correctly ligated plasmid.
Now you pick that colony, and get 98% of the colony with the empty vector and 2% of the colony with the correct plasmid.
Now you grow that up in in liquid culture or on another plate and it grows as "one" colony since they're both resistant to the antibiotic.
For PCR, 2% of that other colony is way more than enough to be the template in your PCR. 35 cycles later, you will get a nice single band amplified from that 2%.
But for sequencing, since you are using M13 which is on the vector, it's going to anneal to both types of vectors. And since the sample is mostly empty vector, that is going to overpower your sequencing reaction and the computer will spit out the final base pair decisions as identical to the vector.
To see if this is the case, you can redo the PCR with both M13 and M13 Reverse (which are both on the vector) and see what you get. If you get two bands, one very small and one the size of your insert, then you know it is contaminated.
I appreciate you. Thank you so much!