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.
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
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.