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

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

2-hybrid libraries

2-hybrid libraries - Protocols and Methods Forum

2-hybrid libraries - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

LinkBack Thread Tools Display Modes
Old 09-01-2005, 02:35 AM
Chris Fisher
Posts: n/a
Default 2-hybrid libraries

I've isolated an interesting cDNA from a human Clontech library that could
only have been made via transplicing from mRNAs originating from 2 different
chromosomes. It seems to be real, but before I follow up on it I was
wondering if anyone with experience in cloning has found cases where similar
artifacts might have arisen in library construction, or of other possible
problems with commercially available libraries. Or perhaps have advice on
how I might tell if it's an artefact of library construction. In this case
the putative exon boundaries don't seem to follow what's been published for
the genes in question, but with splice donor/acceptor sites being pretty
degenerate in humans I don't really know if this is a big red flag or
not--the whole cDNA encodes a rather interesting open reading frame.

Thanks, Chris

Reply With Quote
Old 09-01-2005, 07:33 PM
Peter Ellis
Posts: n/a
Default 2-hybrid libraries

[Only registered users see links. ] wrote:

Chimeric clones are far from uncommon. I've seen this many times
before, especially when making subtracted libraries. The PCR-Select
cDNA subtraction kit from Clontech involves a restriction digest of the
double-stranded cDNA, followed by a blunt end ligation step to add on
the adaptors. At this point it's easy to ligate two separate inserts

If it's a subtracted cDNA library, this is most likely the cause. If
so, it's quite likely (but not 100%) that there will be an Rsa1
restriction site (GT'AC) at the join between the two.

The easy way to confirm if it's real or not is to design an RT-PCR to
amplify across the join, and see if you can recover a clone from the
same tissue that was used to make the library. If not, it's almost
certainly a library construction artefact.

Even if you do confirm that there's a genuine transcript matching your
clone, I'd be wary of claiming trans-splicing - there's more than you
think lurking in the "heterochromatic" holes in the genome sequence,
and jumbled/recombinant loci are among them.

Peter Ellis
Reply With Quote

2hybrid , libraries

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
P[acman] BAC libraries P[acman] Resources Drosophila Forum 0 04-21-2009 07:05 PM
one hybrid system Zahur Muzna Yeast Forum 0 07-09-2007 12:08 PM
BAC libraries Roberta Bergero Protocols and Methods Forum 0 05-18-2007 02:47 PM
2 hybrid library amplification and plasmid isolation problems Gregory Finnigan Yeast Forum 0 08-17-2005 05:04 AM
Self-assembling Devices At The Nanoscale: A New Hybrid Technique Could Lead To Mass-produced Chips With Molecular-scale Structure Do Wah Ditty Physics Forum 1 08-06-2003 11:20 AM

All times are GMT. The time now is 07:08 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11926 seconds with 16 queries