Amount of DNA to run on gel - Be it from PCR or from DNA extraction steps (Jun/18/2008 )

Dear all,

Does it matter how much DNA (usually in ng or ug) we ran in our gel? For most of my work, I always use 1 uL of loading dye (diluted) and 5 uL of my sample and works out fine all these while. Just in case it is not scientific, how do we actually calculate the "weight" of the DNA in a PCR product or extraction?

Thanks.

-dreamchaser_jc-

QUOTE (dreamchaser_jc @ Jun 18 2008, 08:26 PM)
Dear all,

Does it matter how much DNA (usually in ng or ug) we ran in our gel? For most of my work, I always use 1 uL of loading dye (diluted) and 5 uL of my sample and works out fine all these while. Just in case it is not scientific, how do we actually calculate the "weight" of the DNA in a PCR product or extraction?

Thanks.

It is convenient, but not scientific at all, to run 5ul without any idea as to concentration (unless you are running it to check the concentration by comparing it against a specific quantity of DNA ladder).

If you are using EtBr gel, you need about 20ng of DNA for it to be comfortably visible under UV. So, you first measure the concentration of your DNA by spectrophotometry, and run 25-100ng on the gel (if you are expecting only one band; multiply that 25-100ng with the number of bands if you expect more than one band).

hth/

-cellcounter-

QUOTE (cellcounter @ Jun 19 2008, 12:32 PM)
QUOTE (dreamchaser_jc @ Jun 18 2008, 08:26 PM)
Dear all,

Does it matter how much DNA (usually in ng or ug) we ran in our gel? For most of my work, I always use 1 uL of loading dye (diluted) and 5 uL of my sample and works out fine all these while. Just in case it is not scientific, how do we actually calculate the "weight" of the DNA in a PCR product or extraction?

Thanks.

It is convenient, but not scientific at all, to run 5ul without any idea as to concentration (unless you are running it to check the concentration by comparing it against a specific quantity of DNA ladder).

If you are using EtBr gel, you need about 20ng of DNA for it to be comfortably visible under UV. So, you first measure the concentration of your DNA by spectrophotometry, and run 25-100ng on the gel (if you are expecting only one band; multiply that 25-100ng with the number of bands if you expect more than one band).

hth/

Thanks cellcounter. But when you mentioned spectrophotometry, I think there is a constant to be multiplied with the absorption value? and at what wavelength it should be? I know its not scientific and want to prepare for the day when these "conveniences" can't be used. Thanks again.

-dreamchaser_jc-

QUOTE (dreamchaser_jc @ Jun 18 2008, 08:47 PM)
Thanks cellcounter. But when you mentioned spectrophotometry, I think there is a constant to be multiplied with the absorption value? and at what wavelength it should be? I know its not scientific and want to prepare for the day when these "conveniences" can't be used. Thanks again.

OD x 50, 260 Wavelength, 260/280, 260/230 ratios for purity.