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.

Genomic library construction troubleshooting

Genomic library construction troubleshooting - Protocols and Methods Forum

Genomic library construction troubleshooting - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

LinkBack Thread Tools Display Modes
Old 03-03-2009, 10:37 PM
Jayakumar, R
Posts: n/a
Default Genomic library construction troubleshooting

Why is the ligase ethanol precipitated? Cant you buy pure ligase from NEB or from some company. And secondly, try not to use CIAP. It can cause more problems than it resolves if it is not completely deactivated. Assuming that after the ligation, the BAMHI sites are lost on the new vector+insert, redigest the ligated mix with BamHI, which should cut only the self ligated constructs (along with a small percentage of Sau3AI inserts which had BamHI sites), and then use the ligated mix for transformation. You will be selecting for more transformed clones in this case. It will be a good idea to do large scale colony hybridization screening with your probe of interest to select for clones that have your inserts of interestthan doing individual plasmid preps. Saves time in screening the library as well.
Wouldn't it be a better idea to use lambda systems from Stratagene which can take larger insert sizes, screen the plaques for your insert of interest and then subclone them into plasmid vectors. Think about it.


-----Original Message-----
From: [Only registered users see links. ] [mailto:[Only registered users see links. ].indiana.edu] On Behalf Of Rosario Díaz-González
Sent: Tuesday, March 03, 2009 7:10 AM
To: [Only registered users see links. ]
Subject: Genomic library construction troubleshooting

Hi everybody,

I'm working on a genomic library construction for 2-h, but I'm unable to get any insert in my vector.

I'm working with pACT2 vector, digested with Bam HI and dephosphorylated with CIAP (also tried with SAP). The insert is a partially Sau3A digestedgenomic DNA, and ligated with different ratios (1:1, 1:3, 1:5). Both thevector and the insert are agarose-cleaned by Qiaex, and the ligase is EtOH ppt.... but all the few colonies I got are empty, w/o any insert in.

Anything I'm missing? Any tip or hint?

Thanks a lot in advance.


Methods mailing list
[Only registered users see links. ]
[Only registered users see links. ]

This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.
Reply With Quote

construction , genomic , library , troubleshooting

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
Genomic library construction troubleshooting Rosario Díaz-González Protocols and Methods Forum 6 03-02-2010 11:10 AM
Genomic library construction troubleshooting Duncan Clark Protocols and Methods Forum 0 03-05-2009 09:17 AM
Genomic library construction troubleshooting shifali chatrath Protocols and Methods Forum 0 03-04-2009 09:31 AM
S. cerevisiae Genomic Library David A Robinson Yeast Forum 0 05-24-2006 06:04 PM
[BBF] News Issue #4 - Recent bamboo developments in Brazil regarding CHINA/BELGIUM Cooperation Joao Paulo Paglione Botany Forum 0 12-21-2003 04:53 PM

All times are GMT. The time now is 01:15 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12243 seconds with 16 queries