Ameya P

A Few Good Men in my lab.

Hi Paridhy,

Welcome to BioForum.

It would be great if you could post some information.

1. Gel picture of your unspecific bands that you are getting.
2. How are you extracting gDNA from patient samples ( Often blood proteins (contaminants) do not let your PCR work. Hence the non specific amplification. Am guessing here that your positive control template is purified with a protocol different from the one you are using for extracting gDNA.

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.

Dr. DoLittle in my lab

Gods must be crazy  in my lab

use it to aliquot PCR master mix.  read it in a pipette instruction manual  (geeky, yes we all are)

Guess Adrian and me are the only ones (so far) who do it....  thus the 2 votes

Home work?

BTW, welcome to Bioforum and the wikipedia black out is over.

Hi all,

This forum did not have new posts, so I thought I would contribute.....

I am not going to b**ch about colleagues at work or my boss. Am just looking for a bit of counselling.

I finished my MSc in Sep 2009, got my degree in 2010 and officially started looking for jobs. I finally got one In Aug 2010 and the plan then was to work for over an year and then head for a PhD.

Now, its over an year and I must start looking for a place, a PI and a good project. But what is troubling me is this

1. Funding for international students is a rare event now . I cannot fund it myself for I still have an education loan to pay back to the bank.
2. I have some experience under my belt and am enjoying my work. So, is it a good idea to leave all this and start off again ?
3.  The number of PhD graduates is continuously  increasing and in another 4 years (if I complete it right on time) would have probably doubled. What sort of prospects do I have then?
4. Schemes like Clinical Scientist Training in UK, Canada etc are interesting but highly competitive. The added factor of being a non-resident is not going to help my application.
5. The biggest question is this: Even if I set aside all this and decide to take up a PhD, I have to look for a PI and a lab. I graduated with a project in Mol Microbiology which I loved. Here, I am working on human genetics and a bit of biotechnological applications like fingerprinting etc. Now, where do I go from here on. To the microbe lab or look for something like cancer genetics (probably for the reason that it still continues to attract funds even in the days of economic depression).

To be very honest, I am not keen on pursuing a PhD here in India. We have great job opportunities and even institutes of great repute, but so far have not been able to find a PI whom I would like to work with. And for me, the project and PI matter and not the reputation of the institute.

So, please let me know what I might have overlooked or am stressing too much on or is just an illusion of mine. Applications for the next year start somewhere now in Oct- Nov. so I have some time. Your suggestions will help me a lot.

Thanks,
Ameya

Massiri,

Looks like the second primer you are using is not very specific. Try and add two to three bases to the primer and it should work out. If you cannot change primers for any other reason, increase your Tm1 from 48 to 50 or 52oC. That should reduce the non specific bands to a certain extent. Hope this helps.

Ameya

I think, unlike medicine, where people get the impression of having supernatural powers, research teaches you to be humble and honest. If clinicians make more money, with superficial knowledge and fail to see it, it reinforces the idea that their thinking is shallow. There are some clinicians that I know who are not interested in research but also strive to get involved in it along with their clinical duties.

Until a few years ago, I would sincerely believe that clinicians are mere parrots, who can do nothing more. I even tried to convince my friends who took up MBBS. But they probably saw something in the field, that could not fascinate me. Now, it does not matter to me anymore. They are registered clinicians now and are proud of their achievements. Probably primers working out during the first trial would not have given them so much joy. But it makes me happy... and that is all that I care about

I cant really do anything.... its just been about 6 months into my job (its also my first one).
So I can do nothing about it. But yes, I would like to know and learn how to negotiate salaries for my future. I cant accept (or afford) low wages throughout my life.  

I think the agarose is not polymerizing properly in this case. Give it some more time for polymerization.

I saw someone mentioning gloves which resulted in contamination in this forum. Use separate pair for gloves to prepare your mastermix and to aliquot it into tubes. Also, get a brand new set of primers (it might be contaminated).

Ameya