leelee

the dye in the loading buffer is a tracking dye. you use it to judge when to end the run. if you are losing the band during the run then it is probably diffusing and not due to the visualization dye. you may be able to see it if you look at the gel on a white background (we put white paper under).

prabhubct, on 03 September 2012 - 08:41 PM, said:

If that's a plate, it's not possible. Also it still only hides the fact, that other samples may be cross contaminated too.

Too complex for me.  I anneal at 55C.

a friend used to use it for viscous liquids. i'm more patient.

Is your target gene GC rich? IF yes, you might consider add DMSO, BSA or Betaine as addictives.
Also, can you describe your PCR conditions and mastermix in detail? It will be very hard for us to troubleshoot for you without sufficient information.

Bioluminescence is completely different from fluorescence.  Things that glow all by themselves are bioluminescent.  There are several chemically distinct types of bioluminescence.  I'd recommend you look at the literature on insect bioluminescence.

Some bioluminescent systems use fluorescent molecules to alter the color of the bioluminescence.  GFP was isolated from a bioluminescent organism, where it converted blue light to green light.

From my experience and comments my supervisor made whilst I was writing up. If you are working exclusively on writing and have strong writing and editing skills then 3-4 months is possible. For anyone with average writing skills at least 6 months writing will be required. Also, you will need to factor into your timing how long it takes your supervisor(s) to return draft's and if they have any upcoming leave or work commitments that may add delays.

As for recommendations on how to write:
Don't try to write the perfect document/chapter the first time. It is more important to just get thoughts and ideas onto the page. Once you have a few pages of notes/comments you will feel a lot more motivated to write than staring at a blank page.

Do your referencing as you go, even if you are just putting notes on the page. It will save you heaps of time in the later stages when you are editing. Also, use a reference manager (EndNote, ProCite, etc.)

Start writing NOW even if you experiments are not finished you can write 1/2 to 3/4 of a chapter without results and then just add the rest later.

And finally when the crushing wave of panic comes (and it happens to everyone) just breathe, step away from the computer and listen to your favourite song/ go for a walk (anything to distract you from your thesis for a few minutes, no longer than 1 hour) and then sit back down and start again.  You will get there in the end.

Good luck!

goyabean, on Aug 10 2009, 10:15 AM, said:

We have been doing qPCRs in a "dirty" room, without any contamination. Negative controls do not amplify anything ever.

So, while good measures are always good, I would not get myself bogged down in intricate planning unless..

1. You are dealing with a project that requires GMP.
2. Your negative controls are giving some positive results.
3. You are dealing with an environmentally abundant target.

Just do the experiment with reasonable measures to avoid contamination, and see if you really need anything more.

Unless your cells are really really precious, don't decontaminate them, just get/make some new ones.  If you do decide to go ahead with decontamination, you will need to characterize the cells after the treatment to ensure that they have not changed in any fashion from the infection and/or the antibiotics.  Use of antibiotics can cause cryptic infections that will play havoc with your experiments.

For decontamination, Wash cells in medium or PBS, use your regular medium, add the antibiotics and replace with fresh antibiotic medium each day.

http://lmgtfy.com/?q=what+is+ELISA

Electroporation is also done on bacterial cells.  The conditions are quite different in terms of the pulse and the shape of the pulse, so check the conditions carefully.  Read Sambrook et al. Molecular cloning: a laboratory manual, for more information.

This is just a (hopefully educated) guess but I think it will be because the optics are probably at the bottom of the plate, so there is no potential interference from the medium when taking the reading.  Also, the attaached cells will be more spread out, rather than spherical, so there will be a larger area of cytoplasm visible for the interaction to be taking place.

HeLa cell line was derived from a very agressive cancer and they are really tolerant so I don't think it might really affect your results if you need them urgently, but it's a kinda unprofesional approach.  If you have time it's better to perform your experiment 100% properly.

Over-treating the cells with trypsin can cause clumping.  Make sure you are only leaving the trypsin on until the cells are starting to lift off, not for a defined time.

in the sentence, the verb is referring to the buffer, not the volume, so, to be grammatically correct, you should use "was" ("...buffer was added...").