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two bands after gel elusion instead of one - (Feb/17/2006 )

Hi everyone
I did ligation of two gel purified products of sizes 836 and 568 bp (after restriction). Ligation was successful as I got the desired band of size 1400 bp, though I got other bands also because of self-ligation. But I did gel elution of the desired band using conventional method and later purification by phenol-chloroform. But when I again checked the band on 0.8% gel now alongwith my desired band (1400 bp) , there was another band of size 10 kb. I do not know from where this band has come. now if I want to restrict this 1400 bp band again should I again do gel elusion. I have already lost too much of my sample.
Thanks for any suggestion.

-promilasharma-

Hi promila,
thats really weird.. i really think there is some handling error which might have happened .. or some contamination in the eluted DNA... the vector backbone size is how much???

-vishy-

There is no vector involved actually because I performed ligation of simply two DNA fragments after amplification.Moreover in our lab we are not using any kind of such vector of size 10 kb. So how can we see contamination of this size of DNA.Should I run 1% gel to check the ligation .

-promilasharma-

Do you have an image of the gel you can post?

Is this 'band' at 10kb actually a band or is it a smear? How have you been able to accurately judge its length? Also, how much sample (mass of DNA) are you adding per well? It might simply be overloading of the gel and aggregation/multimerisation of your product (or other smaller ones) which prevents migration.

-Doc_Martin-

Hi Doc_Martin

Thanks for replying. I do not have image of the gel. But this band was quite clear in the gel because I compared it with the marker band which had the uppermost band of 10 kb. I loaded only 2 ul of the sample on 0.8% of the gel so I do not think there is any aggregation of product but there might be one possibility that because I eluted DNA manually by conventional method (crush and soak) so there might be some gel particles also in the finally eluted material which might hinder the mobility of DNA. Am I right?

-madni-

Sorry in the last reply I did not mention how much DNA I loaded. Actually I loaded only 100 ng in 0.8% gel.

-promilasharma-

Quite possibly. The simplest way is to buy a gel extraction kit (e.g. QIAGEN) in which you melt the agarose first and then bind your DNA to a column. You can repeat the process to ensure all gel contaminants are removed. They're not too expensive if you buy the small kits.

What mass of DNA do you estimate you have in the 2 ┬Ál you load?

How bright is this band and how do you visualise your gel? EtBr in the gel or do you stain after electrophoresis?

-Doc_Martin-