help w/ ecor1 contam!

[Sunil Shaw]

Mandalaparthy (padmmv00 at ac.usfca.edu) wrote:

: Dear Netters,

:  This may seem like the most bizarre thing, but the Eco R1 enzyme and
: buffer we have seem to be contaminated leading to DNA degradation problems
: repeatedly in our lab. We are using absolutely sterile tips, tubes and
: distilled water.If any generous soul out there has any clue as to what may
: be happening I would be really grateful!!!!!!.Even the brand new stuff is
: degrading absolutely good batches of both plasmid and lambda gt10 dna
: preps. I am so frustrated and would really thank anyone who has any help
: or suggestions.

: THANKS IN ADVANCE>Please e-mail to Padmmv00 at ac.usfca.edu.

: Padmaja Mandalaparthy.
: Frustrated Grad student
: USF, CA.

We had this problem with EcoR1. Turns out that the buffer was bad and was 
causing star activity - a huge ladder of bands. Make fresh buffer, with 
fresh DTT. I think that the DTT had degraded due to freeze thawing. 
Anyway, fresh buffer + old enzyme gave us the expected bands.
Good Luck.
Sunil
skshaw at husc.harvard.edu