Sequencing results failed - (Jul/31/2009 )

I was wondering if anyone would help me with my little problem.

I sent my plasmids for sequencing to check if my insert had been inserted correctly. When the results came back, they said the PCR failed and I did not have any results. I was wondering what could have happened? I get cells when i perform transformation and when I do the over night grow up, the tubes go cloudy and I get a good plasmid concentration after I extract plasmid with spin mini prep kit. What could be wrong??? Im stumped.

Thank you in advance

So many things could go wrong. . . not sure what "failed" means. It could be that your sequencing primer doesn't work - have you tried it before? It could be you have too much or too little template DNA for your sequencer to be happy. Did you dilute it correctly? Could be that you did not remove all the ethanol from your plasmid prep? Is your primer on the plasmid or part of the insert? Ideally, you'd like one on the plasmid before the insert . . . can you talk to whomever did your sequencing for you to see if they can give you some suggestions as to why it might have failed?

aw thank you so much for replying.

The primer I used had been used before by other colleges and it worked for them and I am using the same vector as them.......The primer is for the vector right before the insert.

Also how do I check if all the ethanol from the mini prep had fully evaporated? Would i just let it stand for an additional 1 min?

Thank you

Ms new scientist on Jul 31 2009, 11:35 AM said:

You could put your tube in a fume hood for a minute. That way it evaporates. Usually the ambient lab condition is pretty stable, and EtOH takes longer to evaporate.

I would suggest you do a digest of your plasmid before sending it for sequencing. That way, if you have two band of the appropriate sizes, you know that the transformation worked.

What kind of plasmid are you using? and how are you screening for inserts?

i had this happen a couple of times with what i knew was good quality plasmid. i found that when i changed to a another sequencing company my results were fine...perhaps this is the case here? i would rather not name and shame company!

are you running the big dye terminator reaction yourself? AS mentioned, using too much DNA template can cause a read failure but so too as using too much big dye terminator. Can you tell us what exactly are you doing? How much reagent are you using. quantity of DNA. Your PCR cycling conditions, tm of primer. What kind of clean up method are you using to clean up the big dye sequencing reaction. We need every detail.

Hi

Thank you for all your replies. My problem is (from the start):

I ligated and transformed my insert in a pmal p2x vector into SURE competent cells.

I get maybe 2 colonies per transformation plate which I then perform Spin Mini Prep from QIAGEN. Following their instructions, I grow the colony overnight in a LB-Carbenecillin 50ug/ml falcon tube. I then purify the plasmid using their kit and one strange thing I noticed was that when I measure the concentration of the plasmid extracted, it is low (50-80ng/ul) but then I send it for sequencing anyway and the sequencing results came back 'failed' with too many overlaping signals on the graph they gave me.

Also another thing I did was to perform colony PCR on some of the colonies and I get many bands (of higher and lower molecular mass than the insert). It is as though the bacterial genomic DNA underwent PCR.

I might try and reduce the number of cycles and increase annealing temperature for PCR but what else can I do? And also why did my sequencing results fail? It seems to be contamination (many overlaps) but from what?

Thank you so much for your help!