TigTigger

Hi I have a query about ordering DNA substrates for NMR...According to literature for a 1:1 ratio DNA:protein I need to titrate 10mM DNA substrate per NMR reaction. Most of the facilities that offer synthesis of oligos produce uM concentrations. Where can I order these quantites of DNA (in the UK), for NMR (I am also assuming that they need to be HPLC purified?)? Any help would be muchly appreciated

We all use different pipettes....but maybe should try new ones. Any thoughts about what it could be if not phage??!!! It's obviously immensely frustrating!!

The overnight inoculations grow fine i.e. inoculating with a single transformant, we then inoculate LB 1/100 with overnight...the cells grow normally for 2+ hours then just as OD595 0.4-0.5 is reached clearing starts and what looks like protein clumps appear in the medium. Earlier today I did a small scale prep (2x500ml flasks), 1 flask lysed before optimal OD reached, other a couple of hours into induction...so something obviously got into that one when I added IPTG!!! The flasks aren't opened at all in the incubator and everything is done using sterile technique. I inoculated the remaining overnight culture with 1/100 dilution of lysed culture, but no lysis apparent!!! verrrrrry odd!!!!

Yup and tried using alternative incubators in other locations...it's most puzzling that the cell lysis doesn't occur in every flask...have a suspicion it's something in the lab environment rather than just isolated to an incubator?

Hello!! Right we have a leetle problem in our lab and I was wondering if any wise folks out there may have an idea or solution...any thoughts at all most welcome.... Something is killing our E.coli...about 2 hours after inoculation the E.coli start to lyse. Initially this happened infrequently so it wasn't really a cause for concern, but now it happens almost every prep. Sometimes 1 or 2 flasks will survive, so I think can safely rule out problems with reagents. We have tried using new flasks, new stocks of antibiotic, new medium, transformants etc etc. I know that this sounds like a phage infection, serial dilutions of the lysed culture onto plates of confluent E.coli showed no bands of clearing one would expect in the presence of phage??!!!! Help!!!!! xx