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Gel Electrophoresis: Help needed


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#1 Thomas Congdon

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Posted 21 February 2014 - 09:25 AM

Hey everyone

 

I'm pretty much doing anything I can to try and fix my Gel. I'm trying to get Drosophila DNA to run through a PCR and have our products show up on a 1% agarose Gel. We're using a Ethanol Extraction to extract their DNA. Here's the protocol we used with our last extraction, PCR run, and Gel set up, as well as a picture of how the last gel turned out.

 

DNA Extraction:

 

2 tubes of 4 flies and 2 tubes of 8 flies were collected. 50 uL of H2O was added and the bodies were homogenized (Or grinded, but homogenized sounds sciency). Another 150 uL of H2O was added to the tubes and were vortexed. Tubes were then placed into a 14000rpm centrifuge for 5 minutes. We transferred the supernatant to a new tube and discarded the old ones. Then we centrifuged again for another 5 minutes and transferred the supernatant to a new tube. Then we added 10% total volume of 3M sodium Acetate 6.0 pH. 2X total volume of 100% ethanol was added. We centrifuged the tubes again for 10 minutes. We then discarded the supernatant and added 500 uL of 70% ethanol. We vortexed the tubes and centrifuged them for 2 minutes. After discarding the supernatant we allowed the tubes to air dry and resuspended in 50 uL of TE buffer. This is where things get difficult for me. No matter what I do I can't get the pellet to resuspend by itself. I've shaken the tube, left in an a shaker over night, hot water baths, and vortexing. I now just take a pipette and mash up the DNA pellet untill all I see are very small chunks.

 

With our DNA ready we use ProMega Green MasterMix for our PCR set and run it through our PCR.

 

I prepare our Gel with 0.5 grams of agar and 50 mL of TAE buffer, 5 uL of Ethidium bromide and I run the gel.

 

If you want me to elaborate on any parts of what I just wrote, I'll be more than happy to clear it up. Thank you in advance!

 

also interesting note, in lane 5 I took up a visible chunk of DNA before running the PCR so I'm not sure if that affected the Gel in anyway.

Attached Files



#2 hobglobin

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Posted 21 February 2014 - 09:37 AM

With this protocol you'll lose a lot or all DNA since you use water and not a buffer in the lysis steps. When you grind the flies, use a buffer containing SDS, EDTA and TRiS at least (better also with Proteinase K). If not e.g. nucleases will cut the fly's DNA while you grind and centrifuge. Also the extraction yield is low as you have no SDS to destroy membranes.

I guess the pellet is then something different mostly (e.g. proteins) but no or only very few DNA at all, which might explain the problems when you try to resuspend it.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#3 jerryshelly1

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Posted 21 February 2014 - 09:40 AM

Has this protocol worked before? Homogenization wouldn't be enough to rupture the cells for DNA extraction. Adding sodium acetate will do nothing for DNA precipitation if the DNA is still contained within the membrane. I would add a lysis buffer to your cells and nix the vigorous homogenization.

 

Edit - Hobglobin beat me to it. Listen to him.



#4 Thomas Congdon

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Posted 21 February 2014 - 09:49 AM

No this protocol hasn't really worked before. My research advisor just handed it to me and said good luck haha.

 

Do you guys have any protocols that make use of this sds, edta, tris, proteinase k buffer?



#5 hobglobin

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Posted 21 February 2014 - 10:02 AM

TNES buffer is a good one, you can google it too.
 
TNES buffer (Tris, NaCl, EDTA, SDS): 
 10 mM Tris, pH 7.5
 400 mM NaCl
 100 mM EDTA
 0.6 % SDS
 
add about 5 uL Proteinase K (20mg/ml stock) for 50 uL TNES and then griding and finally some incubation time e.g. at 55°C for two hours or 37°C over night (the longer the higher the yield).


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#6 phage434

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Posted 21 February 2014 - 04:58 PM

If all you are doing with the DNA is a PCR reaction, then the less you do to the DNA the better. I would grind in the TNES buffer then add very small amounts (1 ul) to a 50 ul PCR reaction. No spins, no ethanol, just add it. You could also try some dilutions. Is there reason to believe the PCR reaction is working?



#7 hobglobin

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Posted 22 February 2014 - 10:22 AM

If all you are doing with the DNA is a PCR reaction, then the less you do to the DNA the better. I would grind in the TNES buffer then add very small amounts (1 ul) to a 50 ul PCR reaction. No spins, no ethanol, just add it. You could also try some dilutions. Is there reason to believe the PCR reaction is working?

But then the buffer have to be without proteinase k. If I saw it right (didn't calculate it), the SDS should be diluted enough that it's not inhibiting the polymerase.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#8 phage434

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Posted 22 February 2014 - 10:55 AM

That's right. No proteinase-K, and small amounts of SDS. Freeze/thaw in TNES would be a good approach, I think. Back and forth several times between warm water and dry ice/ethanol or LN2. Or a bead beater (vortex with small glass beads).



#9 Thomas Congdon

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Posted 24 February 2014 - 08:52 AM

So are you saying it will be alright for me to grind the flies in this buffer, with no proteinase k added, and just take the grinded solution into a PCR mix and run it?



#10 hobglobin

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Posted 24 February 2014 - 09:43 AM

So are you saying it will be alright for me to grind the flies in this buffer, with no proteinase k added, and just take the grinded solution into a PCR mix and run it?

Yes this would be the quick and dirty approach...it surely can work. The DNA might be not as long lasting as when you clean it more thoroughly and for other applications than PCR I'd also purify it more.

SDS can be as mentioned above critical too, but either the concentration is low enough or use phage's advice.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#11 phage434

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Posted 24 February 2014 - 09:45 AM

I would supplement your grinding with a few cycles of freeze/thaw if that is convenient. But yes, you don't need a pure DNA preparation to do PCR. And you don't need much DNA.



#12 Thomas Congdon

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Posted 25 February 2014 - 12:23 PM

So I made the TNES buffer and when I stored it over night in the fridge it turned into a milky color. I didn't think much at first but after another day it started to solidify into a jelly. should I be worried?



#13 phage434

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Posted 25 February 2014 - 12:52 PM

Did you autoclave the SDS? It is not necessary and will do weird things. Other than that, I can't imagine what is happening.



#14 hobglobin

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Posted 25 February 2014 - 01:37 PM

Well the SDS will precipitate at low temperature in the fridge but usually not a jelly then...perhaps something other also precipitated? Anyway warm it up and stir, if it goes away it was SDS..

It's not necessary to store it in fridge btw


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#15 Thomas Congdon

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Posted 25 February 2014 - 01:51 PM

I took it out of the fridge and it's starting to clear up a bit. I also ran another gel, I'll post a picture of what it looks like tonight since the computer in our lab doesn't work with gmail for whatever reason. I had 6 wells running and only one of them had some sort of cloudy product in it. I'm not sure if it's actually DNA or some sort of contaminant. Either way, I'm contemplating whether I should try to clean the DNA I got a few days ago currently sitting in TNES buffer now.






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