The Lab Monkey Thread of Joy and Despair Watch

=gabriel=
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#361
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#361
(Original post by Bekaboo)
As in just add too much loading buffer when you load the well? It's possible I guess... Although I'd be surprised if it was that and I've never done it before... still, new gel required methinks.
For me it's usual too little buffer. As in if you add 10X instead of 5X of a loading buffer to your purified DNA in elution buffer or milliQ. But it could be other things. Running another gel sounds like a good move.
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Athena
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#362
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#362
PubMed is down. I might cry!
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Cirsium
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#363
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#363
Hmm second gel run, looks exactly the same. Currently waiting for buffer solution to defrost so i can run a new PCR. Grrr
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Athena
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#364
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(Original post by Bekaboo)
Hmm second gel run, looks exactly the same. Currently waiting for buffer solution to defrost so i can run a new PCR. Grrr
I can offer no advice other than: nice nail varnish.

Could you extract the band and Nanodrop it or something like that?
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Cirsium
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#365
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(Original post by Athena)
I can offer no advice other than: nice nail varnish.

Could you extract the band and Nanodrop it or something like that?

Hehe yeah nice colour isn't it? I got it to match my hair Shame it chips like a *****...

Not sure nanodropping will help - it'll only tell me how much DNA there is after all.

Never mind, I've got a series of control PCRs on at the moment - no enzyme, just genomic DNA and water and I'll run those two and my newly done regular PCR against some genomic that hasn't been in the PCR machine....

Hopefully this will all make sense soon...
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=gabriel=
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#366
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#366
(Original post by Bekaboo)

Hehe yeah nice colour isn't it? I got it to match my hair Shame it chips like a *****...

Not sure nanodropping will help - it'll only tell me how much DNA there is after all.

Never mind, I've got a series of control PCRs on at the moment - no enzyme, just genomic DNA and water and I'll run those two and my newly done regular PCR against some genomic that hasn't been in the PCR machine....

Hopefully this will all make sense soon...
Sounds like you could have genomic DNA in there if you were using it as your PCR template, sometimes you even get bits of genomic DNA contaminating a miniprep if you vortex the lysed cells (not that you should do that).

Also I find PCR sometimes gives amplification products that have no clear rational explanation. For example, in colony PCR you often get amplification, albeit weak, even from lysates that have NO copy of the gene you are interested in at exactly the right size. This is very strange. I got that several times while doing a Quikchange on a long yeast plasmid. Background genomic amplification wouldn't give you the right amplicon length...
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Cirsium
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#367
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(Original post by =gabriel=)
Sounds like you could have genomic DNA in there if you were using it as your PCR template, sometimes you even get bits of genomic DNA contaminating a miniprep if you vortex the lysed cells (not that you should do that).

Also I find PCR sometimes gives amplification products that have no clear rational explanation. For example, in colony PCR you often get amplification, albeit weak, even from lysates that have NO copy of the gene you are interested in at exactly the right size. This is very strange. I got that several times while doing a Quikchange on a long yeast plasmid. Background genomic amplification wouldn't give you the right amplicon length...
Which is why I went for the "no enzyme" and "just genomic DNA and water" controls. Neither amplified anything. Still the same ridiculous band in the 'real' PCR, this time with two bands of a more normal size as well. Hmmm think this one is just gonna have to be sequenced
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Athena
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#368
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We've got a tissue culture contamination problem in our lab AGAIN (I lost eight freshly thawed cell lines which were master stocks before Christmas...), even though we totally decontaminated the hoods, the incubators, the centrifuges, the water bath, all the pipettes and every surface before AND after Christmas (during which time we had nothing growing). the next step is (peroxide?) fumigation Where the hell are the little buggers coming from?!
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Athena
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#369
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I have a nasty Trizol/chloroform headache from RNA extractions Now I see why we use an RNeasy kit - I don't know how the PhD student who sent us the samples can stand to do a whole Trizol protocol!
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Cirsium
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#370
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Really? Never had an issue with TRIzol...
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Athena
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#371
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(Original post by Bekaboo)
Really? Never had an issue with TRIzol...
Do you use it in the fume hood? Because we weren't (the fume hood is too poorly lit to be able to see the RNA fraction :rolleyes: )
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Cirsium
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#372
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(Original post by Athena)
Do you use it in the fume hood? Because we weren't (the fume hood is too poorly lit to be able to see the RNA fraction :rolleyes: )

We don't have a fume hood :ninja:
Always just used it on the bench. Just the regular manufacturer mini protocol in 1.5mLs yeah? Never had a problem.
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Athena
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#373
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(Original post by Bekaboo)

We don't have a fume hood :ninja:
Always just used it on the bench. Just the regular manufacturer mini protocol in 1.5mLs yeah? Never had a problem.
No, samples arrived in Trizol, to which we added chloroform and then did a hybrid RNeasy/DNase protocol. It could well have been the chloroform - I googled "chloroform headache" and there are some forums with people talking about getting headaches from even a very small amount used in a fume hood!

Usually I use the tissue culture hood instead, especially for anything smelly :ninja:
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Cirsium
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#374
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(Original post by Athena)
No, samples arrived in Trizol, to which we added chloroform and then did a hybrid RNeasy/DNase protocol. It could well have been the chloroform - I googled "chloroform headache" and there are some forums with people talking about getting headaches from even a very small amount used in a fume hood!

Usually I use the tissue culture hood instead, especially for anything smelly :ninja:
Ooooh okay. Well Trizol contains phenol, so it could well be that. I used to get Phenol headaches doing DNA extractions. Although chloroform does really get some people. Someone spilled a whole load in Kat's lab and didn't tell anyone and they all started going wooooozy
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cpchem
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#375
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Chloroform can be a bit of a problem, but only really if you're dealing with reasonably big volumes (>100 ml) without adequate ventilation, I've found.
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=gabriel=
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#376
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After a day's worth of hard work and a litre scale extraction, I isolated the original reaction solvent (Dowtherm A). Product buggered off somewhere. Sigh.
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Athena
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#377
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Argh, I've been waiting three weeks for some antibodies to come from France, and the purchasing department haven't even sent payment :pinch: I should have just paid for them on my debit card, it's only €230 and I could have claimed it back quicker than this :bawling:
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Cirsium
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#378
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#378
For those who love the PCR song

:love: http://www.youtube.com/watch?v=ysMJuXJmom4
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Cirsium
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#379
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#379
Long range PCR worked!!!! Long range PCR WORKED!!!!!!
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Athena
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#380
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#380
(Original post by Bekaboo)
Long range PCR worked!!!! Long range PCR WORKED!!!!!!
:party:
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