Growing BAC cultures successfuly

Laura F Marek lmarek at iastate.edu
Tue Mar 31 11:10:13 EST 1998


In article <suglueck-ya023080002403981133250001 at news.ucs.indiana.edu>, suglueck at indiana.edu 
says...
>
>I need tips on growing liquid cultures of cells transformed with BAC c
>lones
>in order to carry out Qiagen maxipreps. The goal is to get as much BAC
> DNA
>as possible, but these bugs are hard to grow and I suspect the copy nu
>mber
>is low. Following the Qiagen protocol (grow a single colony in 5 mL
>LB+Chloramphenicol @37°C for 8H, then in 100 mL of the same overnight)
>produced very little DNA. The BAC in question is pBeloBAC which has
>chloramphenicol resistance. 
>
>Thanks,
>
>Susan Glueck

Susan,

Hopefully someone has responded more promptly to your post, but in 
case not, I have some experience with BACs.  pBeloBAC is very low 
copy - 1 per cell usually.  I have tried a number of different ways to
increase yield and settled on doing standard alkaline lysis minipreps
with some modifications including, using 7.5 M ammonium acetate for 
Soln III (tip from TIGR) and centrifuging at about 7000 rpm after the
 5 min incubation on ice after adding Soln III.  Super goes to a clean 
tube and is respun or, if the DNA is for sequencing, the super is 
extracted once with phenol chloroform and then again with chloroform 
before being precipitated with alcohol.  A 50 mL LB chlor (richer 
broths seem only to increase bacterial DNA contamination) overnight
 culture grown for 18-22 hrs at 35C yields enough DNA to digest an 
aliquot with NotI and do a CHEF to check concentration and insert
size, plus do 8-10 direct sequencing runs (now that our facility
 is using the "Big Dyes") and a few miscellaneous Southerns. 
My experience of trying to bind BACs to any kind of a matrix and
then wash the DNA off was not a positive one - inserts are too
 big etc.  The Qiagen preps are useful, however, to prepare the vector
 to use for cloning.

Laura

Laura Fredrick Marek
Agronomy Department
Iowa State University
lmarek at iastate.edu

   




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