pGEM v pTOPO PCR products for sequencing.... Help! - (Nov/08/2006 )

Hi,

We use an automated sequencer (Beckman Coulter CEQ) to sequence PCR products directly. We have successfully troubleshooted various glitches, and now follow a fairly standardized lab protocol for sequencing:

~ Clone PCR product into pGEM or pTOPO (or other vector that contains flanking M13 primers)

~ Amplify from clone with M13 primers; plate clean or EXO-SAP, and verify a robust single product of correct size

~ Sequence with M13 primers on Beckman automated sequencer (using the DTCS Quickstart master mix recommended from Beckman Coulter)

This approach usually works well. When it doesn't it is often because the M13-amplified PCR product wasn't strong enough or clean enough to sequence.

But recently we have switched from using pTOPO as a cloning vector, to using pGEM as a cloning vector, and we are seeing problems with our sequences. The M13 amplification still works very well, but the sequencing is marginal at best. Side by side runs show pTOPO clones sequencing well, and pGEM clones not sequencing very well.

Now, the vector sequences immediately downstream of the M13 primer (but immediately upstream of the insert) will be different between these two cloning vectors (pTOPO v pGEM.) Could this short stretch of vector sequence be introducing difficulties for the polymerase?

Has anyone else seen these sorts of issues in automated sequencing? Is pTOPO known for being more reliable for sequencing? Would something like smallish changes in GC content affect sequencing runs? Etc? Do you have any suggestions for other things to try?

-Patty

Footnotes:
1. Yes, we've tried sequencing with other primers including the original PCR primers. This has not particularly helped.
2. Yes, we've tried sequencing directly off the plasmid clones. That hasn't worked yet. The Beckman sequencer seems to be fickle, with regards to primers and other conditions, which is why we have generally standardized to the method outlined above.

Help!

Another Q: has anyone seen differences in sequence quality between M13 forward and M13 reverse?

umm... this maybe not relevent to this topic as I have not used pGEM....but yes, I think there is some difference between M13F and M13R primers in terms of sequencing... for some reason the M13F seems to give better quality sequence... longer reads and less likely to fail. I made this observation on pBlueScriptSKII and to a much lesser degree on TOPO.

I have not made actual counts... so this observation is my "feeling" on the matter.