BamHI and HindIII double digest - driving me mad... (Aug/31/2009 )

Hi,

I am trying to digest pcDNA 3.1(-) vector with BamHI and HindIII but the digest does not seem to be working. Vector is 5.5kb and when both enzymes successfully cut I should have 4.5kb fragment and 1kb fragment. I have tested each enzyme individually and both cut the vector once and produce a nice band at 5.5kb. However when I do a double digest or a sequential digest I get the same result each time - one band around 5.5kb which suggests plasmid is just linearized. Looks identical to band that is generated when cut with each enzyme individually. Any idea why I dont get the 2 bands?

Here's my protocols:

Double digest in one tube
Hind III 1ul
Bam HI 2ul
Roche Buffer B 5ul (Have also tried in NEB buffer 2)
Vector 1ul (1ug/ul conc)
H2O 41 ul

Total volume: 50ul

Have left at 37C for 2hrs and have also tried 37C overnight - same result

Sequential digest
Hind III 1ul
NEB Buffer 2 5ul
Vector 1ul
H2O 43 ul

Total volume: 50 ul - @ 37C for 2hrs. Then clean up with Qiagen PCR purification kit.

Then to 30 ul elute add
Bam HI 2ul
Buffer 2 5ul
BSA 0.5ul
H2O 12.5 ul

Total volume: 50 ul - @ 37C for 2hrs. Run on gel & still only 1 band at 5.5kb.

Any idea's as to why I am not getting 4.4kb band and 1kb band?

Thanks in advance.