I sent a gene i recovered from gel after I amplified the gene from the plasmid extracted from metagenomic library by degenerative primers to a sequencing company but the sequencing failed several times although I sent high quality pure DNA recovered from bright thick gene band . sequences sometimes was successful but gave a sequence that i queried for it on NCBI but i did not find relevant gene sequences. And the company told me that the reason may be the degenerative primers that is not specific. And how to solve this problem?
ATTACHED DOEN THE PHOTO OF THE DNA BAND THAT I RECOVERED AND SENT FOR SEQUENCING.
To me the 2 samples close to the ladder look like 2 bands so you may be getting 2 overlying sequences. Can you clone the band and sequence clones to get good sequences
The amplified DNA is overloaded on the gel. 10 and 30-fold dilutions of the DNA would quickly resolve the question as to how many products or one pure amplified product are present.
Thanks alot professor Paul and professor John Paul Rutland John - Collins for your answers . I will purify more and make sure i am sending identical bands next time
You could try sub-cloning your fragments, TOPO cloning can be used to capture either blunt or TA overhang fragments. The pTOPO vectors have M13F and M13R primer sites. These work great both for PCR verification of cloning and for sequencing.
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