BAC for transgenic mice
Posted 08 May 2011 - 10:36 PM
Do anyone know the standard practice for this? Do I need to ensure every single basepair is correct via sequencing or is whatever that i'm planning to do sufficient to give the green light to start working with the BAC?
Thanks a lot!
Posted 09 May 2011 - 12:39 AM
Hope that helps?
Posted 11 May 2011 - 05:32 PM
Hope that helps?
Hi, thanks for your reply. The region of interest we're looking at is 20+kb, so i guess i should at least sequence that. I'm not really familiar with RFLP. I know for RFLP should do restriction enzyme digestion, run through agarose gel, and do a southern blot right? But for southern blot you need a probe, how do you design that probe/how to decide which region to use for the probe? Also for the probe, is it going to be radiolabelled or is there non-radiolabelled form like biotinlyated? Is it as good if I just do a restriction enzyme digestion map, in other words, do the digestion and run through gel via pulse-field (can't do normal gel electrophoresis right?)?
As for purifying the BAC, do you use kit or any particular method? I found the miniprep protocol on bac.chori.org and was wondering if that is sufficient because i did see other protocols online like using CsCl gradient.
Sorry for the many question as i'm really new with handling BAC.
Thanks so much.
Posted 11 May 2011 - 08:28 PM
All this said, our parental BAC (from which all mutants are made) has been fully sequenced by our lab after it was constructed.
Did you make the BAC yourself or get it from someone else? Do they have the sequence?
If you constructed the BAC yourself, it may be worth getting it sequenced to begin with.
Posted 11 May 2011 - 08:32 PM
If I'm just screening during BAC mutagenesis, I'll do a basic mini prep, but once I'm finished, I'll do up a bigger prep and extract using the midi-kit from Machery-Nagel. I'm pretty sure we use the NucleoBond Xtra Midi kit, but I can get the kit cat number for you if you like?
Posted 12 May 2011 - 09:00 PM
For the kit, i'll just google myself. So since you use the kit i assume that it has no issue with shearing the bac?
Also, do you know if i need to sequence the bac, either only around 20kb or the full 200+kb, what is the best method? I read that i could do shot gun sequencing whereby the bac is randomly cleaved to shorter length, ta cloned to plasmid, and sequenced using primers specific for the plasmid. I was wondering if you know of better and easier method. The size i'm looking at is too small to consider next generation sequencing right?
Edited by krystle, 12 May 2011 - 09:11 PM.
Posted 24 May 2011 - 08:43 PM
And no, no issues with shearing of my BAC with that kit.