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
- - - - -

gel extraction problems with the new kit from Macherey&Nagel

gel extraction

  • Please log in to reply
5 replies to this topic

#1 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 14 November 2012 - 02:18 PM

Since Macherey&Nagel has changed their NucleoSpin Extract II kit, we have problems cloning. We have isolated the problem to the gel extraction part. Did any of you observe any problems there?

Andreea

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,379 posts
227
Excellent

Posted 14 November 2012 - 02:21 PM

I'd like to convince you to avoid gel purification. Why are you doing it?

#3 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 14 November 2012 - 02:28 PM

weeell: I have been having the same thoughts lately. However, since I am kind of the person figuring this thing out for the entire department, let us not have to explain to 30 people that the cloning how they used to know it is wrong Posted Image
So a classical cloning protocol in our lab:
1 - PCR insert + insertion of restriction sites + DpnI digest ON + PCR clean-up kit + RE 15 min to 2 h + inactivation + PCR clean-up
2- Midi prep the vector + RE as above + inactivation + gel extraction
3-ligate with T4 DNA ligase for 15 min to 1 hour at RT or ON at 16oC
4-transform in XL1Blue

I have tested everything. Let's assume that I have huge amounts of evidence that it is the gel extraction step aka all the people that had vectors digested in the freezer have successful cloning; people who do Topo or Gateway cloning as well. But for specific vectors you really have to use the classical way.

Andreea

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,379 posts
227
Excellent

Posted 14 November 2012 - 02:49 PM

I would do this with this technique:
1) PCR insert, Ampure bead cleanup, RE digest (with DpnI), heat kill
2) PCR vector, Ampure bead cleanup, RE digest (with DpnI), heat kill (this can be done ahead of time, carefully)
Choose a vector with different resistance than your insert. Possible if you have a broad range of prepared vectors.
We have our standard vectors in amp, kan, tet, chlor.
3) Ligate 15 min on the bench.
4) Transform to DH10B

For some reason, people think there is a reason not to PCR vectors. But it is by far the easiest and most reliable way to prepare low-background vector backbones. Usually, you don't even care about the accuracy of the vector -- if it conveys resistance and carries your insert, you're fine.

Note that this scheme works well for multi-part cloning for making more complex assemblies.

Just say no to gels and columns!

#5 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 14 November 2012 - 02:57 PM

I did this for exactly what you said: multi-part cloning e.g. producing new vectors; but now I really have to make it possible for the people to use the old fashion way that used to work. And I am wondering whether is the kit (which would be such a not ok thing) or the gel staining bath- we use Gel Green. It might be the bath since no matter how many times I try to explain to people not to leave gels overnight or to refresh it regularly, they do not get it.

For myself, I can do what you suggested. However, for some of the clonings I do at the moment I cannot have another resistance vector. I thought about this; or ccdB or sacB... but for these insect cell expression vectors I do not have another alternative other than insert from Amp template to Amp vector.

Andreea

#6 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,379 posts
227
Excellent

Posted 14 November 2012 - 05:54 PM

It can pay off rapidly to engineer a few different resistance plasmids. Your cloning can be done in any vector with any resistance, and you can transfer to an amp vector in a final stage (using the PCR technique).





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

©1999-2013 Protocol Online, All rights reserved.