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
- - - - -

Transformation or Ligation problems

Transformation Ligation TOP10 T4 DNA Ligase pCRII

  • Please log in to reply
4 replies to this topic

#1 JessiBelli89

JessiBelli89

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 07 February 2013 - 02:19 AM

Hey everybody,

i just graduated as a bachelor of science in biology. Now i'm working in the labratory as a labratory assistent and got the task to prepare in situ probes. But I have already trouble with the first steps :(

I ligated a 1kb fragment into pCRII vector and transformed One Shot TOP10 cells with this ligation mix. But unfurtunately I just got many blue colonies and a few white colonies which doesn't have my 1kb insert. I tried it several times but I got always the same result.

Can somebody help me with my problem? I would be very pleased because I don't have a clue :(

Sorry for the bad english..and thanks for lots of answers :)

Jess

#2 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
33
Excellent

Posted 07 February 2013 - 07:29 AM

Can you provide more information. You are using a plasmid with an X-gal gene so you can immediately eliminate all white colonies from your selection procedure. Did you verify the presence of your insert within your plasmid before transformation. Do a PCR with primers specific for both insert and vector. You can also confirm by digesting your vector with a couple of RE to obtain unique sizes.

#3 JessiBelli89

JessiBelli89

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 07 February 2013 - 09:00 AM

Hey, at first thanks for ur answer. The plasmid I'm using carries a lacZalpha fragment with a MCS. Therefore integration of my insert causes a frameshift and all my positive colonies are supposed to be white. Afterwards I isolated the plasmids and digested the construct to get a band for my Insert and the vector.

I tried to check for the insert befor transformation by digestion with the same enzyme (EcoRI) I am using for analyzing my clones.
Unfortunately the concentration was to low to visualize clear bands on the gels. I just saw very slight bands for the plasmids but definitely nothing for the insert.

But returning to ur answer. I will definitely try the version with the primer if available tomorrow. Otherwise I repeate the digestion protocol with a higher amount of my ligation mix.

I will also proceed some control reactions explained in the kit. I will see what's coming out of this.

I think too that the problem is the ligation. What would u suggest do optimize my ligation. I proceeded ligation with a 300bp fragment before and ligation and transformation worked fine. Do u think increasing the concentration of the PCR-Prod could do the job?

Thanks for the reply, kind regards Jess


#4 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
33
Excellent

Posted 07 February 2013 - 03:21 PM

I honestly don't know. Double check your plasmid and Insert concentrations. Maybe adjust your vector to insert ratio or try a different ligation condition. Is your insert blunt or sticky ended?

#5 JessiBelli89

JessiBelli89

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 08 February 2013 - 01:44 AM

I am using a vector with single 3' deoxythymidine (T) residues and the PCR-Product is amplificated with DreamTaq Polymerase which suppose to add single deoxyadenosine (A) to the ends of the PCR-Products. Therefore the PCR Product is allowed to ligate with the vector.





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

©1999-2013 Protocol Online, All rights reserved.