Growing BAC cultures successfuly

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

In article <suglueck-ya023080002403981133250001 at>, suglueck at 
>I need tips on growing liquid cultures of cells transformed with BAC c
>in order to carry out Qiagen maxipreps. The goal is to get as much BAC
>as possible, but these bugs are hard to grow and I suspect the copy nu
>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. 
>Susan Glueck


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 Fredrick Marek
Agronomy Department
Iowa State University
lmarek at


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