November 15, 2011 at 3:42 pm #15689NmrParticipant
I wish that u can help me to come over this problem !
I had done PCR for 16s rRNA gene using colony PCR technique and i used and then i sent my pcr products to the 1st base company without purification in order to do sequencing … they sent me back and they said that i have primer dimer with my products to all my samples.!
So, plz guys what should i do to prevent the primer dimer again and is there any solution that the 1st base company still can doing to give me a good sequence result without repeating the PCR again. , Bcoz dont have time :0
However; this is the second time that i did , the first one that i sent the give me bad sequence with the reverse one so i could not blast my sequence to identify mt sample.
anyway here the primers that is used…
Forward “5’-AGA GTT TGA TCC TGG CTC AG-3’
Revrese 5’-ACG GTC ATA CCT TGT TAC GAC TT-3’
and the following is what the company said to me
1. Kindly advise if you would like to:
a. resend the mentioned samples; OR
b. proceed with the order (we suggest this only if you acknowledged that the DNA quality could compromise DNA sequencing quality to some extent); OR
c. Take up our Gel Extraction / PCR Clean-up service, described below at a separate charge.
(i) PCR Clean-up:
A clean-up method to remove dNTPs, short oligo fragments, mineral oil, enzymes from a PCR reaction product. It does not eliminate unspecific bands and primer-dimers. The quality of the cycle sequencing result is very much dependant of the priming efficiency of the sequencing primer.
(ii) Gel Extraction by General Agarose:
Isolate and purify the target PCR fragment from unspecific bands, primer-dimers and smear products. It would reduce the yield of the DNA greatly and is only recommended if you are not able to clone the purified fragment prior cycle sequencing. The quality of the cycle sequencing result is very much dependant of the priming efficiency of the sequencing primer.
By the way after the company purified my samples i got a very clear bands ( i couldnt upload the Pic )
finaly, i need ur suggestion and ur tips.
November 15, 2011 at 6:52 pm #108128canalonParticipant
If you really are in a hurry, you can go for c (ii). You can also do it yourself and clean whatever is left of your PCR and resend it, if you have some. Otherwise I would suggest that you redo a nice clean PCR for maximum yield, gel purify the samples and send that instead. Even playing with the conditions of the PCR to optimize results could be a good idea. But of course this is time consuming….
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