Trouble with miniprep- DNA contamination

Steven Cohen sbc4 at PO.CWRU.EDU
Sat Sep 14 20:49:12 EST 1996


At 08:45 PM 9/14/96 GMT, you wrote:
>

>However, there is contamination.  My vector is ~2.7k and I see a strong
>band at approx. 12kb that is a brighter than my vector band.  We have
>puzzled over whether this could be 'denatured supercoiled' plasmid or possibly
>RNA or chromosomal DNA, but it seems resistant to specific restriction
>endonucleases.  Additionally, if I miniprep DH5-alpha cells without
transforming,
>I still get the contamination band.  These cells do not have any other plasmid
>already in them.
>
I vote for contaminating plasmid or chromosomal DNA. The fact that non
transformed cells give the 12 kb entity eliminates  'denatured supercoiled'.
This would run quite a bit less than 12 kb for a 2.7 kb plasmid too. I would
borrow some DH5-alpha cells from someone else (preferable from someone in
another lab) and try a transformation or blank mini prep. This will show if
your cell stock is contaminated or if its related to you mini prep. 

Good Luck

Steve




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