leelee

Probably she is just not there. She has not checked her emails and is totally oblivious to the fact that an event like this has occurred.  

Innocent till proven guilty

Curtis,

I can understand your frustration and the amount of time and energy you must have spent on getting the plasmid to work. But I think  you are being too harsh to her. Agreed, she should have replied by now,  but like leelee said, she might not just have seen your mail. She is just a caretaker of the plasmid, so its very likely, that she does not know much about the plasmid. She just sent you the sequence, she has on her record (which could have been wrong in the first place). So, she is not really to blame.

And why are you belittling your own work/ lab for such a small thing. You do not really know how things are in Oxford/ Cambridge, unless you are actually there.... (the grass looks greener on the other side)

Besides, if she really wanted you to have the wrong plasmid, she could have sent you one, why go through the whole rigmarole of sending you an carefully edited sequence. Or not send you one at all.

Yes, you have been at the receiving end of all this, and we really do not know what you have gone through, but at least give her one chance of clearing the air on this isssue. Questioning her education qualifications is a bit too harsh....  

While you have mentioned that you have lowered your annealing temperature, how sure are you that 56 is the optimum temp for the PCR. have you tried anything lower? What are the Tm for your primers?

Just for additional info, what are you planning to do next, with the PCR product?
A gel picture of the low PCR yield would be of great help to all of us here.

Search PubMed for the T2 cell line AND MHC class I and you should find some helpful papers.  They are back in the mid-1990's.  Let me know if you have any trouble tracking them down and I can give you specific papers.

I think the idea is a nice one, however, to some greater or lesser extent, many PhD programs require that you act as a teacher (typically demonstrating labs and tutorials) during the process, so there is already this component to the degree.  If you are interested in teaching at a school level, as opposed to university, I think this course would be good (based on the description), but it is unlikely to secure you a faculty position in a university - first off because if you are going to be doing research and teaching, they will employ you based on your research skills largely, and second if you are going for a teaching only position, then you need to be specialized in the topic that you will be teaching already.

You should make sure that the fridge is one that doesn't have a freeze/thaw cycle built into the system - usually known as a frost- free freezer - they warm up the area to keep it free of ice so the temperature is much less stable than in a normal freezer.  It isn't so important for fridges, but many fridges still have the function built in to ensure that areas around the cooling equipment remain free of ice.

You probably want to maximize the number of shelves and avoid butter warmers and vege bins.

I Am Legend in my lab

From Dusk Till Dawn in my lab

Doctor Who in my lab

Gods must be crazy  in my lab

This looks as if it is an artifact of the gelling of the agarose.  I suspect that you have changed the way you are mixing or cooling the agarose before pouring the gel, and that some of it is setting more rapidly than other parts, leading to non-uniformity of the gel.  Make sure the agarose is well mixed and sufficiently warm before pouring the gel.  The solution should be crystal clear before pouring the gel.

We store primers at -20, and don't worry about freeze thaw.  We keep DNA ladders at room temperature for many months when stored in TE, with no evidence of degradation.  TE is your friend.

Yeah - EtBr was (possibly still is) used as a prophylactic and treatment for sleeping sickness in cattle at about 1 mg/kg (that's milligrams/kilogram - just so you don't think its a typo) with no observable effects.

I may run two gels if I am doing both, after loading fresh buffer.  But I do not trust the buffer left over in a gel box others have used.  I even wash the gel box out after I'm done (probably the only time it ever gets washed).

and for the announcement of the results of the replication of the experiment to prove its repeatability, they'll use baskerville? Perhaps then I'll believe it