Sequence without primer

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camembert
camembert's picture
Sequence without primer

I have a good sequence of my target DNA after PCR and sequencing, however my primer sequence which I search for as a reference point of my unknown DNA was not there. I found a part of the plasmid sequence and immediatelly my unknown sample.

How is this possible?

labrat
labrat's picture
can you give me some more

can you give me some more details?

are you pcr'ing from a plasmid, then sequencing from the pcr product, or pcr'ing, then cloning, then sequencing direct from the plasmid?

is the sequencing primer you're using the same as one of your pcr primers?

I couldn't quite get a clear picture of exactly what you've done from your question.

natascha
natascha's picture
I'm ligating a PCR product

I'm ligating a PCR product into a pGEM vector. with this vector i perform a PCR with Primers on the vector next to the insert product. After PCR I sequence. When I have a normal Sequence Im looking for this vector primer to locate my desired sequence. at this clone I couldn't find the primer but the sequence was correct because I had an idea of which specie I was clonuing and t gave me the same sequence. I'm sequencing the d2d3 region of nematodes

camembert
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The anwer of Natascha is

The anwer of Natascha is actually from camembert I was accidentally anserred unther a wrong loggin on our PC.

vanishing
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Created multiple accounts?

Created multiple accounts?
;-)

vanishing
vanishing's picture
So you are using primers for

So you are using primers for PCR that amplify your PCR product and for sequencing, you use a primer that lies upstream (in the sequence of the vector)?

What type of sequence method are you using?
Fluorescently-labeled nucleotides?

camembert
camembert's picture
NO I don't multiple accounts

NO I don't multiple accounts Natascha is my girl friend who's also a scientist. I was working from home when I answerred.

camembert
camembert's picture
yes I'm using a vector primer

yes I'm using a vector primer that is upstream.

I use the ABI PRISM Big Dye Terminator Cycle Sequence Ready Reacton Kits Of Applied Biosystems.

They 're using the following fluorescent markers a 6-FAM donor and a dRhodamine acceptor dye.

labrat
labrat's picture
I'm still not quite clear

I'm still not quite clear what you're saying you do. From your second post, it sounds like you sequence from a pcr product that has been generated from a plasmid with insert. Is that correct?

Or, are you sequencing direct from plasmid, with your sequencing primer lying just outside one of your PCR primers?And when you sequence you get the non-PCR primer vector sequence, and then the insert, but not the vector sequence that corresponds to your pcr primer?

cloner
cloner's picture
It sounds as if you are

It sounds as if you are trying to see the sequencing primer, which is not possible. At best, one can see the bases immediately adjacent to the primer, but readable sequence usually begins 10-30 bp downstream from the primer.

Scot E. DOwd Ph.D.
Scot E. DOwd Ph.D.'s picture
Yeah unless you are looking

Yeah unless you are looking at the actual trace file you will likely not see the primer as it will be screened out for poor quality. when looking at the trace the primer will appear as a cluster f. SO the previous answer is correct