3 replies to this topic

[j47027]

I am having a problem getting a straight ladder on my DNA gels. The bands are curving (like a broad "U"). I use 2log ladder. I have tried using lower voltages in different percentage gels and the results are the same.

Gel volumes i use are 50ml, 70ml and 100ml at 0.8%, 1% and 1.2% (for all gels). I use the 2Volts/cm for 10mins at the start then crank it up to 5Volts/cm for the rest of the run.

NEB recommend to use 1ul ladder/1ul 6X loading dye/4ul water per lane. I make up the original loading dye stock 100ul ladder/100ul 6X loading dye/400ul water when I get the 2log ladder to the lab. (I add 6ul of pre-made stock to well)

EtBr is added to agarose when agarose is melted (when its cool enough) and gels are made in TAE. the lower MW bands are sometimes straight but the high MW bands are always curved.

Does anybody have advice for me on this??
It really annoying having to show somebody my gel image when its looks bad.

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[jeff kwak]

do your samples look like that too? or is it just your ladder(s)? to be honest, i wouldnt worry too much. you dont have to publish the ladder anyway, but it seems like the problem is the ladder itself. i would borrow another ladder from another lab and run it next to each other. see if you get any different results than they do. let me know where you got your ladder, so i wont order the same one.

my best,

jeff kwak

[j47027]

jeff kwak, on 03 March 2013 - 02:26 PM, said:

do your samples look like that too? or is it just your ladder(s)? to be honest, i wouldnt worry too much. you dont have to publish the ladder anyway, but it seems like the problem is the ladder itself. i would borrow another ladder from another lab and run it next to each other. see if you get any different results than they do. let me know where you got your ladder, so i wont order the same one.

my best,

jeff kwak

I should have put an image up of my samples but yea my samples can look like this too..(i dont have any pics on this laptop)..I got the ladder from New England Biolabs but I have purchased new stocks of the ladder also..I was sure it was voltage that was doing this or tht there was too much ladder being loaded..When I load little amounts of DNA the samples appear nice and straight..If anyone can post a picture of their "2log ladder" from NEB and tell me what their conditions are I may be able to reproduce their results. Yea its not the publications thats the problem its just showing my supervisor or whoever that it can get annoying when comparing band sizes.

[mdfenko]

it looks to me like the samples have started to migrate into the side walls of the sample well prior to or just after starting the electrophoresis (before all of the sample could enter the gel).

how long do the samples sit in the wells before starting the current?