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DNA electrophoersis ladder very blurry


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4 replies to this topic

#1 philman

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Posted 02 November 2010 - 07:36 AM

Recently my DNA reference ladder (Invitrogen 1kb Plus) has been giving me a very blurred smeary result, rather than the distinct bands I am used to and used to get. This only happened after I made the most recent aliquot, although I have tried to make a new aliquot since. This also happened when I tried to make another aliquot using someone elses DNA ladder to see if it was just mine that was contaminated. I find it very strange as I used to get very nice bands!

When making aliquot I take 100ul of the DNA ladder from the freezer, and add 20ul of the 6x loading buffer (Promega 6x Blue/Orange loading buffer)

I have just realised when typing this that that is far too much DNa ladder to be using and tht I should prob be using a 1:10 dilution of that, but does anyone else have any ideas?

#2 bob1

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Posted 02 November 2010 - 03:02 PM

degradation.

#3 phage434

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Posted 04 November 2010 - 02:29 AM

This sounds like what happens when gels are made with water instead of buffer. Is it your gels rather than your ladder?

#4 shivasankari

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Posted 18 November 2010 - 06:01 AM

There could me more than one reason : Are you getting your other bands properly? If not, the problem might be because of the gel. When you are adding too much of the ladder, you get a thick streak. And try to stop the gel half - way , because EtBr runs in the opposite direction from that of the DNA, so in case you run it for too long, the EtBr could have as well passed into the buffer. Check for contamination as well.Buffer pH cud also be an issue!

hope it helps

all de best

Recently my DNA reference ladder (Invitrogen 1kb Plus) has been giving me a very blurred smeary result, rather than the distinct bands I am used to and used to get. This only happened after I made the most recent aliquot, although I have tried to make a new aliquot since. This also happened when I tried to make another aliquot using someone elses DNA ladder to see if it was just mine that was contaminated. I find it very strange as I used to get very nice bands!

When making aliquot I take 100ul of the DNA ladder from the freezer, and add 20ul of the 6x loading buffer (Promega 6x Blue/Orange loading buffer)

I have just realised when typing this that that is far too much DNa ladder to be using and tht I should prob be using a 1:10 dilution of that, but does anyone else have any ideas?


Cheers

Shiva

#5 Kikuyu

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Posted 27 April 2011 - 04:23 AM

You are using way too much ladder.

For 100ul 1 kb marker we use:
10 ul 1 kb marker
25 ul 5xBJ
65 ul ddH2O

For 100bp we use only 5ul (and 70 ul water).




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