1 reply to this topic

[moorele]

ignore this post! made a mistake!

Edited by moorele, 31 August 2009 - 08:45 AM.

[Dr Teeth]

moorele, on Aug 31 2009, 12:09 PM, said:

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.

In pcDNA3.1(+ or -), both BamH1 and HindIII are unique sites found in the the multiple cloning site.  BamH1 site is only ~15 bp up/downstream of the HindIII site (- or +).  Why are you expecting a 1 kb band? You are probably getting good double digestion and seeing the expected 5.5 kb band.  You won't see the very small fragment that is excised from the plasmid.