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DESPERATE, PLEASE HELP!!!! simple gel problem!

DESPERATE, PLEASE HELP!!!! simple gel problem! - Agarose Gel Electrophoresis Forum

DESPERATE, PLEASE HELP!!!! simple gel problem! - Agarose Gel Electrophoresis Forum. A forum to discuss electrophoresis staining, troubleshooting, and development of films for DNA and RNA gels.


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  #1  
Old 07-15-2009, 02:36 AM
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Default DESPERATE, PLEASE HELP!!!! simple gel problem!



i have some problems I am very worried about

I had isolated some RNA and made cDNA, then when i measured the concentration of my cDNA it was insanely high (around 2000 ng/microlitre) but my boss said it was okay and i could go ahead with the PCR, he just told me to dilute it.

so i diluted it with water so that every microliter had about 100 ng. i went ahead and did my PCR.

now on my gel, nothing showed up. i ran it again, this time with a ladder and i also ran my cDNA to see if that would show up.

i ran a 0.8% gel and my ladder was completely streaked through, some bands were kind of visible but not nicely defined, and my cDNA row, there was a clump at the top of the gel, near the well, but it looked like it was streaking too. again, nothing showed up for my PCR samples, but at the very bottom of the gel, past where the dye stain was, there was a clump of stuff visible, for both PCR rows.

my question: is my cDNA streaking because the concentration was too high? this is what i think it was. i put in 2 microliters which was around 4000 ng, that's wrong right? can i use the diluted cDNA (i diluted in water)? if so how much? 100 ng?

and secondly, why is my ladder streaking like that? what can cause a ladder to streak? and how do i fix it?

and thirdly, what is the clump at the bottom of my PCR lanes?!

i am in desperate need of help, please please please help!
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Old 08-07-2009, 02:45 PM
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Default Re: DESPERATE, PLEASE HELP!!!! simple gel problem!

If your ladder is messed up than it must be a problem with a gel. Just prepare fresh TAE, fresh gel, and run a ladder alone or with a plasmid you know is not degraded.

Once you solve the gel problem, the other ones will either go away themselves or you will be able to define them more clearly
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Old 08-07-2009, 08:23 PM
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Default Re: DESPERATE, PLEASE HELP!!!! simple gel problem!

Can you show us the photo of your gel so we may have more clear view of the problem and try to help?
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Old 12-15-2009, 05:18 PM
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Default Re: DESPERATE, PLEASE HELP!!!! simple gel problem!

If its streaking and if your cDNA concentration was strangely high, the cDNA conversion may not have worked and you might have amplified tons of non-specific bands from contaminated gDNA and primers that are not specific enough. Good luck!
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Old 01-13-2010, 02:53 PM
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Default Re: DESPERATE, PLEASE HELP!!!! simple gel problem!

firstly i agree with andriy, you should run the gel one more time again. if you think u load too much DNA don't worry just dilute it. as there are many wells so u can do a 1/5 dilution. I would suggest u change the buffer with fresh one and make sure ur gel is properly solidified.

as aga said, if we can see the gel would be best. especially the one u re-run again.

yes, as lavenderrgirl said it, your cDNA is very high. Do you quantitate it from purified samples or direct from pcr product?

opsy... its been long time since u asked. maybe u can tell us have u solve the thing? thanks!!
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Old 05-10-2010, 12:20 PM
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Default Re: DESPERATE, PLEASE HELP!!!! simple gel problem!

The concentration you are saying is too high for cDNA, though I dont know your RT-PCR method. Run your cDNA at various concentration, make sure that your spec machine is correctly equilibrated. The bottom bands in your PCR samples might be the unused primers perhaps (cant tell without a photo). Did you run rour RNA samples in a gel. Were the subunits clear?
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