Help explain my DNA bands smearing

Knepper, Marc A MKNEPPER at NMU.EDU
Mon Sep 25 17:18:08 EST 1995


>j140474 at singnet.com.sg(jene) wrote:
>>Hello, recently I did a gel electrophoresis on genomic DNA isolated from
>>Arabidopsis. The samples were restricted with MluI, EcoRI and SAU3A.
>>I expected to see distinct bands of DNA but ended up with smears in all
>>my lanes. No bands can be made out at all. The markers came out all righ.
>>
>>What is the smearing due to? Was it caused by multiple site restriction?
>>Can I solve the problem?
>>
>>Anybody who can help please post back a reply to this Newsgroup. Thanks!
>>
>
>Dear Jene,
>
>At a guess your problem is that the DNA is not digested enough and so shows up
>zyme/DNA ratio or simply cut longer (at least 4 hours for 5-15ug DNA) or both t
>
>Liz.
>
>
>j140474 at singnet.com.sg(jene) wrote:
>>Hello, recently I did a gel electrophoresis on genomic DNA isolated from
>>Arabidopsis. The samples were restricted with MluI, EcoRI and SAU3A.
>>I expected to see distinct bands of DNA but ended up with smears in all
>>my lanes. No bands can be made out at all. The markers came out all righ.
>>
>>What is the smearing due to? Was it caused by multiple site restriction?
>>Can I solve the problem?
>>
>>Anybody who can help please post back a reply to this Newsgroup. Thanks!
>>
>
>Dear Jene,
>
>At a guess your problem is that the DNA is not digested enough and so shows up
>zyme/DNA ratio or simply cut longer (at least 4 hours for 5-15ug DNA) or both t
>
>Liz.

Either:

1. The gel is overloaded and you've got way too much DNA in there, or
2. Digestion was incomplete, or
3. The DNA is degraded.

In case of:

1. A good amount of DNA to use for a standard minigel is about 1 ug;
anymore and you can end up with an unreadable gel.  2. Do as suggested
2. Do as suggested above and use more enzyme. Mg++ can also be
supplemented in the reaction, in the form of MgCl2, which is a cofactor
of restriction enzymes and when depleted can severely decrease activity
of the enzyme.
3. Purify more DNA, which can be a pain in the butt, depending on what
you've got.

Good luck, I hope this helps.

Marc Knepper
Northern Michigan University




More information about the Methods mailing list