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
- - - - -

No sequencing of DNA methylation

DNA methylationSequencing failure

  • Please log in to reply
6 replies to this topic

#1 Tati GM

Tati GM

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 14 August 2014 - 03:38 PM

Hi, I need help...

 

I was able to sequencing good sequences of converted DNA, my workflow is DNA purification, converting DNA (Kit) ,  specific PCR, cloning, PCR confirmation of colonies, plasmid extraction and send the plasmid to sequencing with specific primers, my samples are in the right concentration and the size is about 300bp. But at begining of year something happend and now my sequence are how the picture show... The `A` is how my sequences are at the moment (bad), the `B` is how some sequences appear, the `C`and `D`show how then were... Good! The good sequences agreed with the reference.

I don`t known what happening, I have read about the use of SP6, I have already tried... I read about the hairpin that can form due to the excess of repeated bases in conversion... The use of more formamide...

Someone could help me??

 

Thanks

 

Tatiana

(sorry forSequence.jpg bad english)



#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 14 August 2014 - 06:01 PM

Are you sending miniprep plasmid DNA from a single colony? What primer are you using? Is it a primer binding to the vector, or to your DNA sequence? This should have little or nothing to do with the converted DNA if you are sequencing a plasmid with a vector binding primer.



#3 Tati GM

Tati GM

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 15 August 2014 - 07:03 AM

Thanks for your reply.

 

Yes I sent a single colonie, I am trying sequencing with T7 primer because I used pgem T.



#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 15 August 2014 - 07:14 AM

This suggests that the problems you have are unrelated to anything except sequencing of the plasmid. You should try control sequencing of a known good plasmid (doesn't matter what it is,  presumably a pGEMT plasmid). It might also be prudent to streak out your "single colony" for single colonies again, to assure that it is not a mixed population. Make sure your DNA submitted for sequencing has the correct concentration. Both too l ittle and too much DNA can cause problems in the sequencing reaction. Make sure your primer is correct, and at the correct concentration.



#5 Tati GM

Tati GM

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 15 August 2014 - 07:34 AM

Thanks again,

 

I`ll check everthing again, I`ll found a control too, but I follow the protocol (150-300ng / DNA) I used Qubit to check, after delivering the samples to sequencing I don't have control about happens... It's worried me... But I can do the process too!?



#6 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 15 August 2014 - 05:39 PM

Since you are having trouble, I would check the DNA concentration on a gel, not with Qubit. Most ladders (such as NEB 2-log ladder, my favorite) have DNA concentrations associated with each band, if you run a known amount of ladder. Compare your band with one of the ladder bands and estimate the DNA concentration. What counts is the DNA concentration of your desired plasmid, not the total DNA concentration.



#7 Tati GM

Tati GM

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 03 September 2014 - 10:16 AM

Hello

The last test: I checked the concentration (I let all the samples around 280ng), I sent a control, duplicate and I used M13 primer foward and reverse. Total of samples 8, only 5 worked, same in duplicate, the result was the same in both. I still do not understand...How I could attach the picture in reply?







Also tagged with one or more of these keywords: DNA methylationSequencing, failure

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

©1999-2013 Protocol Online, All rights reserved.