Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Molecular Biology Techniques
Register Search Today's Posts Mark Forums Read

Molecular Biology Techniques Molecular Biology Forum. Includes forums for common molecular biology techniques.


cloning problem

cloning problem - Molecular Biology Techniques

cloning problem - Molecular Biology Forum. Includes forums for common molecular biology techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-23-2013, 10:35 AM
Pipette Filler
Points: 83, Level: 1 Points: 83, Level: 1 Points: 83, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default cloning problem



I have a problem when trying to clone a 1kbp gene into a vector. The vector is about 8kb. I PCRed the gene, which works beautifully. Then I digested both the insert and vector using XhoI and BamHI. The DNA was gel purified. Everything seems OK.
However, after I ran the ligation and used the ligation mixture to transform E. coli cells, I did not get a single colony! I did my ligation using Roche rapid ligation kit. Since I did not get colonies, I went back to repeat the ligation experiment and checked the ligation mixture on a gel. I also did a control experiment with both insert and vector, as well as the ligation buffer, but omit the ligase. On the gel, I found the control mixture contains both the linear vector and insert, as I expected. For the mixture with ligase, I saw a smeared band with high molecular weight (higher than the linear vector) and both the bands corresponding to the linear vector and insert disappeared. Therefore, I concluded that at least the ligase works and both the insert and vector can be ligated into something, though I cannot say they can be ligated into the product I want. However, I still have no colonies, even use ligation mixture that I clearly proved to contain some products with above method. I also set a ligation reaction with only the vector. Normally, two vectors should be ligated together and this should give you some colonies (though these colonies are useless for cloning purpose). However, I still have no colonies. Now I start to suspect the competency of my cells(DH5alpha) and even change competent cells to Agilent XL-2 ultracomp cells and, I also did a positve control using intact vector during the transformation, which leads to lots of colonies. Therefore, the cells seem to be OK.
Right now, I do not have any more ideas on how it should work. Do you have more ideas how to trouble shoot this problem?
Still no colonies……with no results………..
Reply With Quote
  #2  
Old 02-25-2013, 04:11 AM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: cloning problem

Is the protein you are trying to clone is poisonous to the Ecoli cells? Thus resulting in no growth.
Reply With Quote
  #3  
Old 02-26-2013, 07:10 AM
Pipette Filler
Points: 83, Level: 1 Points: 83, Level: 1 Points: 83, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: cloning problem

No, I check it with empty pLVX vector only......it is still able to transformed and the colonies are good.

There might be some inhibitors present and I am not able to solve it.
Reply With Quote
  #4  
Old 02-28-2013, 12:41 PM
Pipette Filler
Points: 83, Level: 1 Points: 83, Level: 1 Points: 83, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: cloning problem

no there is no such problems.........those protein will not express.......
Reply With Quote
  #5  
Old 03-02-2013, 02:41 AM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: cloning problem

[Only registered users see links. ]

Check the link.
Reply With Quote
Reply

Tags
cloning , problem


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
ligation-free cloning Maximilian Haeussler Protocols and Methods Forum 10 05-28-2012 12:55 PM
DNA Cloning Protocol moleculardude Molecular Biology Articles and Protocols 2 08-05-2007 02:31 PM
BioInfoMan:reduce your working time on molecular cloning by 80% gaxl Bioinformatics 2 08-15-2006 12:31 AM
Problem cloning in pGEM T easy antaine Protocols and Methods Forum 1 09-13-2005 12:42 AM
FW: SDS-PAGE problem Deanne Bell Protocols and Methods Forum 0 03-05-2004 05:27 PM


All times are GMT. The time now is 05:04 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14008 seconds with 16 queries