PhD support group?

misskool

cupid, yours sounds worse than mine. We've got the whole protocol set up so we know it works, I'm sure it's my samples which no one has worked with before so we are going for the bucket science technique next week of massively overloading the gels and crossing our fingers.
The only thing I can think of for your Western is that your sample buffer might have been contaminated, have you stained the gels with Comassie to make sure you still have the protein? If you do, then check that your transfer is ok with Ponceau. 2-mercapto does go off (although it already smells off!!) so if you've had the SDS+2-mercapto buffer for a long time, time to make new ones.

Dr.Lou

Hiya, Graduated last July with my PhD (Engineering). Know how hellish it can be at times especially with experiments failing...time running out....supervisor changing (in my case 3 times)...etc. Anyone need moral support just PM me too!

On the other hand looking back I really loved it too, and glad I finally got it!

Just looking for a job now....it's really depressing, very rarely I see anything that is suitable here (S.Wales).

ffeindadifyr

dumpy wrote:

I work as an AL for the OU and it is great but I don't know whether I could have done it whilst I was a PhD Student. It's a lot more time consuming that it looks especially the first year you work and it's not that well paid for the amount of work you do.

How much more work is it than that they advertise. i.e. one of the courses says 5hrs a week - how much more than this is actually required?

Advice would be gratefully received :beer:

crazyscientist

Drfluffy - i know that western blotting into an early grave feeling!
Cupid_stunt - PM me with your protocol and problems if you like.

I too am just finishing second year of PhD. Its not going too badly (touch wood) but i know i have a year of hellishly hard work ahead if i want to finish on time (which i do, obviously!!)

Currently trying to write a paper.. what a lovely way to spend a friday evening :-(

misskool

Join the club, I am picking samples and procrastinating.
Be happy that you've got a paper to write, my work is still in pieces!

DrFluffy

cupid_stunt wrote:

And my western blotting hasn't worked for about 7 months now. Grrrrr. Any trouble shooting tips DrFluffy?

What are you blotting for, and what blocking (Milk, BSA or somethng else) and washing solutions (TBS, TBST, other?) are you using?

crazyscientist

misskool and cupid_stunt - have you checked you have enough protease inhibitors in your samples? i use a cocktail of aprotinin, leupeptin, antipain and PMSF at the moment which seems to keep them stable. just thought if you have seen them before and now then have gone then that could be a solution? Definalty agree with checking transfer with ponceau. Also, if you wash membranes between incubations with TWEEN then do some washes without it just before you try to visualise the bands (dont know why this works but it did for me!)


as for the on-the-side working issue, ive done the usual undergrad demonsrating too. supervisor not happy about it in 3rd year though and so im effectivly taking a pay cut this year

DrFluffy

The thing I had difficulties with was the writing style required for a thesis. I'm fairly good at writing papers and being succinct, so the long drawn out prescriptive thesis style came as a shock!

My advice is that the last thing you should write is your introduction. Start with your materials and methods (if science), then your results chapters, and conclusions. Only then do you really know what you have to introduce...

crazyscientist

DrFluffy wrote:

My advice is that the last thing you should write is your introduction. Start with your materials and methods (if science), then your results chapters, and conclusions. Only then do you really know what you have to introduce...

sounds like a good plan

DrFluffy

crazyscientist wrote:

misskool and cupid_stunt - have you checked you have enough protease inhibitors in your samples? i use a cocktail of aprotinin, leupeptin, antipain and PMSF at the moment which seems to keep them stable. just thought if you have seen them before and now then have gone then that could be a solution? Definalty agree with checking transfer with ponceau. Also, if you wash membranes between incubations with TWEEN then do some washes without it just before you try to visualise the bands (dont know why this works but it did for me!)

I also wondered about phosphatases... If looking for phosphorylation, the last thing you should block with is milk (contains buckets of phosphatases), but it seems that this is a common problem.

I always used protease inhibitor tablets - call me lazy! At least you could ensure fresh, uncontaminated stocks...