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without RE site in PCR product


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5 replies to this topic

#1 Junvn

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Posted 23 August 2013 - 03:47 AM

I've got trouble in cloning and sequencing.

 

I amplied fragment of gene I wanted (PCR) with 2 primers, and then check length of PCR product by gel agarose. I sequence PCR product, with reverse primer. I think forward primer is also sequenced in PCR product, this means sequence of PCR product also have forwand primer's sequence, but not have. Of course, primers design to clone, so they have restriction enzyme site. But PCR product is sequenced without restriction enzyme site, why? I dont explain.

 

Please help me! Thanks.



#2 phage434

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Posted 23 August 2013 - 09:36 AM

Your sequencing results will depend on the fragment length. If it is less than, say, 600  bp, you should easily see the end of the fragment. You may see it up to about 900 bp. The last few bases are typically poorly resolved, and there is often a final "A" called which is not really there. The forward primer will be at the end of the sequence, and will be the reverse complement of the primer sequence you originally used. The RE should be present in that sequence. If you have longer fragments, you will not see the end of the fragment by sequencing from the opposite end, and will need a primer to the middle of the fragment to see the end sequence. Sequence after about 700 bp or so will degrade, so you might see poor seqeuencing in that region.



#3 Junvn

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Posted 25 August 2013 - 12:02 AM

Thank you very much. the length of fragment is about 754bp, and the end of fragment on chromatogram by sequencing are not good, in other words, reverse complement of  forward primer are poorly resolved (after 710 bp ).

I wonder what I will do so that I exactly know sequence of that region. In fact, 754 bp are not so long to design middle primer


Edited by Junvn, 25 August 2013 - 12:07 AM.


#4 phage434

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Posted 25 August 2013 - 09:53 AM

It should be very easy to design a primer about 500 bp into your existing sequence read which will very nicely sequence the end of your 754 bp fragment. I would not design the primer any closer than about 100 bp from the end of the fragment.



#5 Junvn

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Posted 25 August 2013 - 11:12 AM

any problem if I design primer closer than 100bp from the end of fragment?...sorry becauz I dont have much experience for primer design

#6 phage434

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Posted 25 August 2013 - 11:29 AM

The best sequence reads are typically from about 150-400 bp after the 3' end of the primer. Dye blobs are common at about 75 and again at about 120 bp, and early sequence 1-30 bp is typically poorly called, although manual reads can often interpret it well. Depending on your sample, instrument, and capillary age, reads start to degrade seriously around 800 bp and become unreadable around 900 bp.






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