Smear in gel electrophoresis (RFLP)

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levadura
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Smear in gel electrophoresis (RFLP)

Post by levadura » Mon Jan 25, 2010 7:12 am

Hi!,

I write you because I need help. Nowadays I'm working with Sacharomyces cerevisiae, I work with the enzyme HinfI to obtain the mt DNA restriction patterns, but when I try to separate on a agarose gel 1% (w/v) (conditions: constant voltage of 60V for 2h; buffer TAE 1x; ), the bans are not visibles correctly due to they have a lot of smear.
I think my problem is the DNA extraction, but I don't know improve it.
Can anyone to help me with this problem?
Thank you very much!!
Attachments
electroforesis_dna_hinfI.jpg
smear in gel electrophoresis (stained with Gel red 1:10.000)
electroforesis_dna_hinfI.jpg (12.49 KiB) Viewed 9265 times

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JackBean
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Post by JackBean » Mon Jan 25, 2010 10:09 am

did you try to load on gel non-digested DNA? Did you try digest by some few-cutting RE?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

levadura
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Re: Smear in gel electrophoresis (RFLP)

Post by levadura » Mon Jan 25, 2010 2:00 pm

Hi!
I only cut with HinfI, I din't try others enzymes. When I loaded non-digested DNA I haven't this problem....I'm lost :( ....
thank you very much!!

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JackBean
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Post by JackBean » Mon Jan 25, 2010 2:28 pm

If you didn't have that problem with non-digested DNA, than the problem is probably not in extraction ;)
Try the other RE (something, what cuts only few times, that is, recognize long sequence) or maybe your reaction except the enzyme
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

anuragpanjabi
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Post by anuragpanjabi » Sat Feb 20, 2010 3:24 pm

Did you put in the Loading dye?

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JackBean
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Post by JackBean » Sat Feb 20, 2010 8:06 pm

I don't think, that loading dye should influence that...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

Lacustris
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Re: Smear in gel electrophoresis (RFLP)

Post by Lacustris » Mon Apr 27, 2015 4:18 pm

Hello,

I also have problem with my electroforesis pictures.
I am doing clasical PCR-RFLP and it offten happen that the resaults are not sharp and I can not be sure about the genotype. Strage is that I for example doing the whole plate and half of samples (on 2 gels) are ok and the 2. half is fuzzy. And as you can see sometimes there are some waves on the band, really don´t know what is that.

Firstly I thought that the gels are warm, but they are not and the electrophoresis run usually about 15-20 min (3,5% agarose + 5uL ethidum bromid or gel red)

For better understanding follow the pictures belowe, plese.

Does anyone already seen or handled with this? Does anyone has any idea?

I'm getting really desperate... :roll:
Attachments
29.15. CXCR 777 b).jpg
Waves on the 1. row
Wrong.jpg
The bad things
Nice.jpg
Nice one

Lacustris
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Re: Smear in gel electrophoresis (RFLP)

Post by Lacustris » Mon Apr 27, 2015 4:23 pm

And the last picture
Attachments
29.15. CXCR 777 c).jpg

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