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
- - - - -

excision of fragment during cloning

cloning

  • Please log in to reply
4 replies to this topic

#1 ajitk

ajitk

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 13 August 2013 - 07:42 AM

Hi,

 

I first ligate 4 different pcr products to make a single insert and then cloned it in mammalian expression vector by using clontech Infusion cloning kit. I used BamH1HF and EcoR1 HF enzymes to cut my vector. Insert has matching vector sequence at both ends with BamH1HF and EcoR1 HF enzymes cutting sites. After cloning we observed sequence of first pcr product and some initial part of second pcr product was cut during cloning reaction. I checked my individual pcr product sequences for having above restriction sites in between but above enzyme sites were not found in any of 4 pcr products.

 

please anybody suggests what is the possible reason behind this. 

 

I will be thankful to you for the same.

 

Ajit K

 

ajitkul1976@yahoo.com

 



#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,468 posts
247
Excellent

Posted 13 August 2013 - 10:54 AM

Did you check the inserts by examining sequence, or by trying to cut them with the enzymes?



#3 ajitk

ajitk

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 13 August 2013 - 10:49 PM

I have checked the insert sequence and found that both the restriction sites are absent. In-Fusion cloning, PCR products are designed in such a way that their both ends have vector matching sequence so there is no need to cut the PCR products by enzymes. I only cut the vector.

 

Thanks and regards,

 

Ajit Kulkarni 



#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,468 posts
247
Excellent

Posted 14 August 2013 - 05:22 AM

Checking the sequence on a computer lets you know if that sequence will be cut. Cutting with the enzyme tells you that the sequence in your tube will not be cut. These are different, unless you have sequenced the DNA in your tube. I'd suggest that your computer sequence is different from your sample sequence, and that the samples can be cut. Of course, other things could also be the problem.



#5 ajitk

ajitk

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 16 August 2013 - 12:34 AM

I did PCR to amplify the gene of interest and purified PCR products get sequenced commercially and then that sequence I checked for enzyme sites, also gene sequence available on Genbank also checked for enzyme site, but not found enzyme site. 

 

Are competent cells  involved in cutting as clontech mention use of certain competent cells. I used competent cells of different company.







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

©1999-2013 Protocol Online, All rights reserved.