June 18th, 2007

Ciariel

Any Advice?

Maybe this isn't the right community for this, so if you can direct me to a different community, that would be excellent.

At the moment, I'm trying to cut out an insert from the pHTT7k plasmid using NdeI and SacI, and then put the insert into a pET28a plasmid. I transform pHTT7k with insert into some competent cells, grow them up, Midi prep, and then digest the pHTT7k. When run on an agarose gel, my digests look successful, so I've been cutting out the band containing the insert, and using a QIAEX II gel extraction kit to get my purified insert. However, at the end of the gel extraction, I'm getting nothing. I've done this 3 times now. I'm following the protocol exactly, I think. I'm making sure all my solutions are at the correct pH.

Is there any tips anyone can offer me as to how to actually give me some success in the gel extraction? Are there alternate protocols I can do, to get my insert purified?

Will SOMEONE talk to me?

Hello, I hope you guys don't mind me dropping in.

I'm a research tech/ de facto lab manager for an MD who does research as well as see patients.

My boss has been out of the lab for over a month.  The Loch Ness Monster has put in an appearance since I've seen Dr. Boss last.  No phone calls, no emails, and no response to the phone messages, pages and emails I send him.  We're running out of grant money and haven't published a paper in three years.  Hell, I could be out of a job and not know it.

Dr. Boss, where the hell are you?  

Also, I've got a student volunteer.  She's 16, wants to go to a "Top 10 School" and become a doctor.  The Top 10 comment irritated me because this U. is not in that catagory - learn some tact kid or shut up!  She doesn't talk much, or even ask questions.  I keep stoping what I'm showing her to ask "Do you understand?  Any questions? Are you sure you got that the first time?"  A little feedback would be nice. 
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