4 replies to this topic

[huangc4]

Hi, I don't know if any one can help.
I am doing the subcloning and just double digested the plasmid (pET-21a) with RE of XhoI and NdeI (both  from NEB).
However, after double digestion (I did it for O/N), the DNA gel showed there was a band on the very top, as if it almost didn't move at all from the loading well. I did the twice and each time saw the same thing. I am just wondering is there anybody else have seen this before? Can any one tell me what this band is on earth?
By the way, using the same way to double digest my pET-28C vector and there is no such a band.

Any suggestion will be deeply appreciated!

Edited by huangc4, 04 February 2009 - 01:51 PM.

[phage434]

Two possibilities:
1) your plasmid prep has substantial genomic DNA contamination, which is carried through to your cut plasmid prep, or

2) restriction enzymes stick to the DNA, retarding band migration.  Have you tried cleaning up the  cut DNA with a column or phenol/chloroform?  You might get rid of this with a heat kill step following digestion at 65C for 20 minutes.

[TanyHark]

huangc4, on Feb 4 2009, 10:20 AM, said:

Hi, I don't know if any one can help.
I am doing the subcloning and just double digested the plasmid (pET-21a) with RE of XhoI and NdeI (both  from NEB).
However, after double digestion (I did it for O/N), the DNA gel showed there was a band on the very top, as if it almost didn't move at all from the loading well. I did the twice and each time saw the same thing. I am just wondering is there anybody else have seen this before? Can any one tell me what this band is on earth?
By the way, using the same way to double digest my pET-28C vector and there is no such a band.

Any suggestion will be deeply appreciated!

if you had run the uncut plasmid control on the same gel, you should have seen (most likely) the same band there too. I am almost sure that is bacterial genomic DNA contamination. You go and do a good plasmid prep again

[huangc4]

Somehow, I am not able to quote your answers using my laptop.
Thanks for your reply.
Sorry that I didn't mention that I did run the uncut plasimid and there was no such a band. So it is not very likely that it's from genomic DNA.

[scolix]

did you run the same amount of uncut plasmid as you were digesting? Could you post a picture of the gel here?