which reagent adds density to DNA? - 3X loading dye (Apr/27/2007 )
we are using 3X loading dye of composition,
5M NaOH 200ul
Bromophenol blue 50mg
xylene cyanol FF 50mg
add dd.water to make up to 100ml
in general, any of , ficol or sucrose or glyserol will be added to the loading dyes to add density to DNA. in this above one which will fullfill this purpose?
our colleague, while preparing used NaOH 5M prepared 6 months ago and while stirring it on the magnetic stirrer he let it for some time, bye mistake some body swiched on the heater of that stirrer. the dye was heated. so, tocool it we put it in ice overnight. morning we loaded DNA samples on 0.8% agarose added with this dye. it ran in different way, which we did't see previously. the xylene cyanol dye ran towarda right side of rhe gel opposite t the gel run and bromophenol blue towards left side i.e. normal way. after halfanhour stained the gel and documented. we got bands, but run was opposit , why?
ficol or sucrose or glycerol
all three can be added to a loading buffer to increase density.
I prefer ficol as bacteria have a harder time growing in it. Thus the loading buffer is less likely to be contaiminated by bacteria. However from a preparative prespective, glycerol is an easier substance to work. Disolving ficol powder is messy and time consuming.
I can only guess what happened with the heated. For the xylene cyanol to run in the opposite direction, towards the catode, the molecule must aquired a positive overall change while boiling in the sodium hydroxide solution. Perhaps it is a mixture of a hydrolysis reaction, which frees up the 2 amine groups on xylene cyanol, and perhaps a nucleaphilic attack to the 2 sulphonyl groups by the 95ml of formamide you have added.
Why are you adding NaOH to your loading dye? Why not just use TE? I've made loading dye with water, TE and SB and there was no difference between them. I just use TE now, with glycerol as the densifying agent.
hi, thank you for ur answer.
the formamide decomposed and the products reacted with the sample.