Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

problem in sequencing of gene

Gene T-vector sequencing

  • Please log in to reply
3 replies to this topic

#1 Huongnguyen.miss

Huongnguyen.miss

    member

  • Active Members
  • Pip
  • 21 posts
0
Neutral

Posted 17 December 2014 - 01:48 AM

i have tried 3 times to send  for sequencing of bacteria gene. this gene i have cloned into T-easy vector. and then i sent it to company for checking the sequence is wrong or correct. but even 3 times , i got wrong all of them. in sequence missed 1 or 2 oligonucleotide at different site in gene.

Does anyone know the reason? i need your help. 

i would so much appreciate your comment


Edited by Huongnguyen.miss, 17 December 2014 - 01:55 AM.


#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,509 posts
254
Excellent

Posted 17 December 2014 - 07:22 AM

Are you looking at the called sequence, or at the electropherogram. Mis-called bases are common at the beginning and end of sequence reads, and about 80=120 bp into the sequence read. Usually these can be identified by looking at the electropherogram.

 

Of course, another possibility is that your organism simply has mutations, which are quite common.



#3 Huongnguyen.miss

Huongnguyen.miss

    member

  • Active Members
  • Pip
  • 21 posts
0
Neutral

Posted 18 December 2014 - 01:16 AM

that is sequencing. i did many genes in genome of that organism ( actinomycete) but just only this gene above 1500bp is problem by its sequencing. i tried to change PCR parameters but still got incorrect sequence, and difference colonies are incorrect at the same site of oligonu. maybe i got mutate from PCR product? 


Edited by Huongnguyen.miss, 18 December 2014 - 05:10 AM.


#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,509 posts
254
Excellent

Posted 18 December 2014 - 06:50 AM

I don't understand. If multiple sequencing results for several colonies of your organism all have the same "incorrect" sequence, then it is not the organisms, or the sequencing results that are wrong, but rather your assumptions. Believe the data, not your preconceptions.







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.