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.


Huge DNA fragment cloning

Huge DNA fragment cloning - Molecular Biology Techniques

Huge DNA fragment cloning - Molecular Biology Forum. Includes forums for common molecular biology techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 01-16-2012, 11:39 AM
Pipette Filler
Points: 102, Level: 2 Points: 102, Level: 2 Points: 102, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2012
Location: germany
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Huge DNA fragment cloning



Hello everybody,

I work with reverse genetic systems for RNA viruses. It is very difficult to clone viral genes, since our viruses harbour GC rich sequences. Complete cDNAs are larger than 16 kb and are usually cloned in pBR322, because a Rob gene is needed for stability. These plasmids are exclusively proliferated in the old E. coli strain HB101 which accepts such ugly bugs, but the bacteria grow very tiny colonies. At the end of the day most of the plasmids still grow very poor , DNA is not easy to preparate and mutations are frequently generated . So my question:
Does anybody know more stable vectors for such large inserts?

Kind regards

crotalus
Reply With Quote
  #2  
Old 01-18-2012, 02:59 PM
Graduate Student
Points: 1,231, Level: 20 Points: 1,231, Level: 20 Points: 1,231, Level: 20
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2011
Posts: 113
Thanks: 0
Thanked 16 Times in 13 Posts
Default Re: Huge DNA fragment cloning

I have grown pretty large inserts of mammalian genomic DNA (20-30 kb) in pBlueScript-II. I know people don't usually recommend high copy plasmids for this kind of stuff but I always use them and I haven't had anything that I couldn't grow at all in pBSII yet.

I use the STBL2 cell line from invitrogen and grow at 30C, sometimes it is necessary to reduce the antibiotic concentration to get good plasmid yields.

Another option would be to use a BAC vector backbone which can hold much larger inserts (100-500 kb).

In terms of mutagenesis I don't typically sequence the entire insert (for my purposes this is not necessary), but the parts that I have sequenced (say approximately 30%) have not had mutations.
Reply With Quote
  #3  
Old 01-27-2012, 10:13 PM
Pipette Filler
Points: 102, Level: 2 Points: 102, Level: 2 Points: 102, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2012
Location: germany
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Huge DNA fragment cloning

Thank you for your suggestions mmorgan!

I tryed several standard plasmids (like pBSII, which is helpfull for the cloning of small RT fragments of my virus). But I was never able to clone more than a single GC rich gene of my virus in such high copy plasmids. Currently, I grow my bacteria at RT with reduced (1/4) ampicillin concentration for 3 days. A single mutation is often fatal for virus replication, so it is a hell of a work to clone a new virus strain (more than one year!). I really like plasmids and am not willing to start BAC work at the moment. I hope that someone from this community has solved the plasmid stability problem.
Reply With Quote
Reply

Tags
cloning , dna , fragment , gc rich , huge , large insert , vector


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
Cloning into pCS2+HA - suggestions needed please genotype Molecular Cloning Forum 0 06-16-2011 08:16 PM
NoeClone: next-generation molecular cloning tool feilr Bioinformatics 0 12-29-2010 05:31 AM
cloning Fab fragment from hybridoma bwax Molecular Biology Techniques 0 07-02-2010 10:49 PM
2.2 fragment kb into 2.6 kb vector - Please help randi912d Molecular Cloning Forum 0 02-25-2010 01:53 AM
BioInfoMan:reduce your working time on molecular cloning by 80% gaxl Bioinformatics 2 08-15-2006 12:31 AM


All times are GMT. The time now is 01:43 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12654 seconds with 16 queries