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Gel electrophoresis problem


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#1 samuel

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Posted 30 January 2010 - 02:50 AM

One week ago, I have no problem on DNA extraction of and the result is as following picture , url= http://img109.images...s/i/goodkq.png/


However, I have encounter some problem on DNA extraction now. I got a different things instead of a good band. The result is as follows: url= http://img64.imagesh...s/i/aftera.png/

I think it is called smearing at the bottom lane, is it? I couldn't find the reason for such changes. I have changes all the reagents and be carefull in every step. But , still i have the same problem for a few times DNA extraction. What is my problem????

Another question is i saw a very very faint line at the upper part of the smearing in second picture. I wonder if it is a band or not?

This problem hunting me for a few weeks already. I hope i can get some comments from yours. Your help is deeply appreciated.

#2 phage434

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Posted 30 January 2010 - 07:39 AM

Your second gel looks great, possibly overloaded with too much DNA. Try running a 10x dilution or smaller amounts in the well. Your marker lane needs more DNA, or the image needs more exposure. I don't see why you think there is a problem. The smeared DNA background you see is likely genomic DNA contamination at low levels. Be careful of excess vortexing of your lysed cells and over long lysis in P2 or whatever lysis buffer you are using. The "band" at the upper end is just limiting resolution of your gel -- anything longer than about 20Kb will run at that length, except for DNA that is so long that it sticks in the well.

#3 HomeBrew

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Posted 30 January 2010 - 10:58 AM

What form of DNA are you trying to isolate? Your first picture looks like chromosomal DNA, and your second looks like a plasmid, or possibly a PCR product. How are you doing your DNA extraction?

#4 samuel

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Posted 30 January 2010 - 02:56 PM

What form of DNA are you trying to isolate? Your first picture looks like chromosomal DNA, and your second looks like a plasmid, or possibly a PCR product. How are you doing your DNA extraction?


I am extract the chicken genomic DNA (By the way, i am using 1kb ladder). So, the DNA band should be in high molecular weight just like the first picture bands. The second gel picture seems out of expected.

#5 samuel

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Posted 30 January 2010 - 02:59 PM

Your second gel looks great, possibly overloaded with too much DNA. Try running a 10x dilution or smaller amounts in the well. Your marker lane needs more DNA, or the image needs more exposure. I don't see why you think there is a problem. The smeared DNA background you see is likely genomic DNA contamination at low levels. Be careful of excess vortexing of your lysed cells and over long lysis in P2 or whatever lysis buffer you are using. The "band" at the upper end is just limiting resolution of your gel -- anything longer than about 20Kb will run at that length, except for DNA that is so long that it sticks in the well.


I suppose i type the wrong title.This is not the gel problem. But, it is a DNA extraction problem. Sorry....

#6 lab rat

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Posted 30 January 2010 - 06:31 PM

hi samuel,

Could you please give your protocol? That will help us help you. :P

Thanks,

lab rat
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#7 samuel

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Posted 30 January 2010 - 07:00 PM

hi samuel,

Could you please give your protocol? That will help us help you. :P

Thanks,

lab rat


sure..

1. Cut out liver tissue of about 0.5X0.5cm in size
2. Wash the tissue with sterile PBS buffer, and leave the tissue in petri dish.
3. Mince tissue into fine pieces and transfer to microcentrifuge tube.
4. Add 500 ul of lysis buffer and then add 50 ul of proteinase k.
5. Incubate the suspension for 1 hour at 55'C water bath.
6. Add equal volume Phenol: Chloroform (1:1) and centrifuge at 13000rpm for 2mins.
7. Transfer aqueous phase (400 ul or 300 ul)into new tube.
8. Add 1/10 3M Sodium Acetate (pH6) and mix.
9. Then add 1 volume absolute ethanol and mix.
10. Centrifuge at 13000rpm for 30 seconds to pellet the DNA.
11. Wash the pellet with 1ml of 70% ethanol twice,
12. Air dried the pellet at RT for 15mins. (Normally, i centrifuge for a second and then pipette out the remaining ethanol to facilitate the drying)
13. Resuspend pellet using 40 ul of nuclease-free water.

#8 phage434

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Posted 31 January 2010 - 06:57 AM

Ignore what I said previously; I thought you were extracting bacterial plasmid DNA.

This protocol should likely say in step 9, add 2.5 volumes of absolute ethanol. 1 volume is not sufficient to precipitate DNA. Perhaps you (or the author of the protocol) were confused by the possible use here of isopropanol, which indeed could be added at 0.8 to 1 volume, but for ethanol you need more.

If I were doing this protocol, I would extract after 6 and 7 a second time with chloroform only to get rid of extra phenol.

You'll never be able to see this DNA on a normal agarose gel, except as a bright band near the edge of the well. You may see contaminating RNA, which this protocol does little to remove. That may be what you see in your gels.




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