Screening of Mouse BAC Clone Libraries - Molecular Biology (Mar/10/2007 )
Anyone has any idea of how I should carry out a screening of the mouse BAC library? My project involves transgenic work using BACs... so I'm looking for a mouse BAC clone with this particular gene in it. Been searching on the internet for 2 days and all I came across are commercial companies offering such services, is that better off using those services or is there a way of doing it on my own online for free?
Typically this would be done with a hybridization probe or with PCR. Which way depends largely on the form of your library. If you have a mixed bacterial clone library, then plating out the clones and doing colony hybridization is the method of choice. If (more likely) you already have picked clones, then colony PCR is the method of choice. With large libraries, it is appropriate to mix multiple sets of colonies (typically 10-20 colonies) and do PCR on the mixed set, which reduces the number of reactions dramatically. You can, for example, pick all of the clones in a row of a 96 or 384 well plate into a single colony PCR reaction. You should definitely test the primers used in such reactions prior to working with the library, and make sure you run positive and negative controls. A robot will be of a lot of help in setting up these reactions.
Thanks for the tip! I'm sure it'll come in handy when I start going into the 'nitty gritty' of molecular cloning.
Cheers & Best!