Clostridial genomic DNA

[C.K. Chen]

In article <33A1CA3A.1CC7 at vetsci.microvet.arizona.edu>,
billing at vetsci.microvet.arizona.edu wrote:

> Does anyone have any thoughts on how to prepare genomic DNA from species
> of Clostridium other than C. perfringens?
> 
> I am trying to prepare genomic DNA from C. novyi type B and C.
> haemolyticum (or novyi type D).  I have no trouble making C. perfringens
> DNA and I can get the other strains to lyse without trouble.  But the
> yields of DNA are low and rapidly degrade (e.g., O/N at 4oC or as soon
> as I try to cut them).
> 
> I am guessing this is due to the action of endogenous DNAses from the
> clostridia, but how do I get around it?  I have tried using
> SDS/proteinase K lysis followed by various combinations of phenol and
> CHCL3 extraction.  I have also tried using a Qiagen genomic prep kit
> which uses guanidine in the solubilization buffer.  This works better
> (ie., the DNA doesn't degrade as quickly), but the yields are usually
> 10-20 fold less than expected.
> 
> Any thoughts or suggestions would be appreciated.
> 
> HJ

We've good lock with the following protocal, which is adapeted from
Verhasselt et. al. 1989, FEMS Microbial. Lett., 59:135-140.

1. Harvest 250ml mid-late log-phase culture by centrigugation at 4C
2. Wash the pellet 1 time of Saline-EDTA (150 mM saline, 100 mM EDTA, pH
8.0) solution
3. resuspend the cell pellet in 9 ml of TE buffer (pH 8.0) and 3 ml 0f 5%
Sarkosyl solution
4. incubate the mixture at 55C for 15 min
5. add 1.2 ml of predigested pronase E (2.5 mg/ml) to the mixture and
incubate for an additional 15 min at 55C
6. add 2.64 ml of a 5M sodium prechlorate solution and 15 ml of a
chloroform:isoamyl alcohol (24:1) solution
7. incubate and gently shake the suspension at 4C for 30 min
8. centrifuge and recover the aqueous upper layer
9. add two volume of cold 100% EtOH and store at -20C for overnight
10. centrifuge and resuspend the DNA pellet in TE buffer containing 20
ug/ml of RNase

C.K. Chen