1 reply to this topic

[alshia]

please help
i have been try to hybridize a 5kb dig labeled probe with high stringency for the last 5 months.
it worked in my training in one lab, now i am doing it on my own with no luck. i am following the instructions that came with kit.

i am using traditonal southern transfers is there any way that i can ensure dna is on the membrane?

Edited by alshia, 13 April 2004 - 03:50 PM.

[leahf]

Hi Alsihia
hope you don't need this by now but just in case...
i don't know which kit you have been using but Roche has a DIG-labelled molecular marker. this is very good as a positive control for your DNA transfer. if transfer works and still no signal. you can check the label of your probe by spotting it on a membrane and detcting (full procedure in Roche;s DIG manual). finnalyy, if your target DNA is large (>5kb), depurinatuion of the gel (several minutes in 250mM HCL) may help.
Luck