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Southern Blot after Gardella-Gel, no Transfer?

Southern Blot after Gardella-Gel, no Transfer? - Molecular Biology Techniques

Southern Blot after Gardella-Gel, no Transfer? - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 04-05-2014, 02:17 PM
Pipette Filler
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Default Southern Blot after Gardella-Gel, no Transfer?



I have some Problems with my Southern Blot after running a Gardella-Gel. I want to Transfer genomic and viral DNA.
I use the positivly charged Nylon Membrane (Amersham Hybond™-N+ Pore size 0.45 µm)

I give you a short sum up of my protocol (Sambrook et al)
Crosslinking for 5 minutes
Depurination: 15 Minutes 0,25M HCl
wash twice with Aqua dest
Denaturation: 2x 15 Minutes 1,5M NaCl/0,5M NaOH
wash with Aqua dest
Neutralisation: 2x 15 Minutes 1M TRIS/1,5M NaCl/pH 7,4
Transferbuffer: 20xSSC pH 7,0

I use a 10cm high paper towel and already tried weights between 2 to10kg. After the night the buffer only draws about 2cm and after staining the gel with EtBr I still see bands. I'm wondering if a Transfer has happend, when the buffer only soaked a few centimeter

Thx
Janine
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Old 05-07-2014, 05:49 PM
Pipette Filler
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Default Re: Southern Blot after Gardella-Gel, no Transfer?

I've tested my transfer with a PCR-Product (500bp) an with my purified cellular DNA. The small PCR-Product has been transferred, but the big virus/genomic DNA hasn't. I prolonged the Depurinationtime from 15 to 30 minutes, but that had no effect. I think that the size of DNA is the problem. Shall I use alkaline transfer with 0,4N NaOH?
Slowly, but surely I become exasperated with this blot. No one in my Lab has experienced with that. ;(
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blot , gardella , gardellagel , southern , southern blot , transfer


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