I've been doing some RF/Gibson cloning, and I am having issues sequencing my plasmids. I performed colony PCR to identify clones with the appropriate inserts. I then grew these up and plasmid prepped them to extract the DNA, which I then quantified using a Nanodrop. When I sent these plasmids for sequencing, the reactions come out as complete trash (ie. a bunch of "unknown" base pairs, or just failed sequences in general). I tried a few troubleshooting techniques:
1) Changing the amount of DNA I prepare for sequencing (according to the guidelines set out by the sequencing company).
2) Using the sequencing company's standard primers instead of adding in my own.
3) Re-streaking the frozen stock and ensuring that I select only a single colony, which I then grow up for plasmid prep.
For all of these, the results still came back the same (ie. bad). I then PCR'd a region that covers the insert, Dpn1 digested it, cleaned it up, quantified via Nanodrop and sent that for sequencing. This time, I got perfect results (ie. everything was exactly as desired).
I am wondering what changed? Why are the plasmid sequencing reactions failing, but a PCR of the plasmid isn't? I have to create 12 clones and it will be quite tedious to PCR all my clones prior to sequencing, rather than sequencing them from the plasmid directly.
Purified plasmid can be a poor template for PCR (sequencing or regular), typically due to secondary structures/supercoiling. You could try linearizing the plasmid and sending that in for sequencing. But it sounds like you have a protocol that works. 12 clones isn't that many. Good luck!
is it possible that your cloned product has 2 of each primer ie the same product is ligated to itself before cloning then the primer produces 2 sequences depending on where the sequencing primer anneals? Are you sequencing with the pcr primers when it fails. Did your second pcr cover the entire insert from plasmid primers or just a part of the insert? Can you post a failed sequence .abi file just in case it has any clues please?
Paul Rutland I guess anything is possible, but I don't think that is the cause in this case. I made sure the primer has only a single binding site in my clone, but also, wouldn't that affect my secondary PCR too? I performed my colony PCR using a forward primer outside my insert and a reverse primer from within my insert, and got the correctly sized fragment. As for my second PCR (the one used for sequencing), the primers I used spanned the entire insert + a few 100bp on either end of the insert, so unless that entire region is somehow duplicated (which seems unlikely), I should only have one product. I can't post the files, but here is how they all pretty much looked like...that or the reaction just dropped off after 500 ish bp.
That is a pity as .abi files contain a lot of useful information. I was interested in your noting that the sequence dropped off at c500bases. If it was a reasonable intensity then this suggests either that there are 2 sequences present ( unlikely as you have sequenced across the insert) or that the primer can anneal in 2 places in the one sequence suggesting either that the same primer exists at both ends of the amplimer or that the primer is slightly degraded and 2 primers of slightly different length are bot annealing and producing a sequence and the result is a messeven though they are both annealing at the same end if the 3' ends of the primers are at a slightly different position the sequence will be messy. This can also happen if the primers are badly synthesised and there is a lot of fail sequence remaining in the primer
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