HELP!! I ruin my Native-PAGE (newbie in PAGE) - My bands seem faint smear (Jun/22/2011 )

Dear expertise..

I am newbie in PAGE and this is my first experience conducting Native-PAGE, particularly.Actually, this is as a preliminary experiment before conducting the IEF. I try to load the genomic DNA in 7.5% Acrylamide gel with the below recipes:

Bis-acry (30%)
4x resolving 15 ml
APS 25% = 120 uL
TEMED = 10 uL

The Anode Buffer = 0.45M Tris/Acetate; 4 g/L SDS. pH: 6.6
The Cathode Buffer = 0.08 M Tris; 6 g/L SDS. pH: 7.1 (actually, the recommendation is added with 0.80 Tricine. But, I omit it coz, my lab doesn’t have the available one )

I utilized the Flatbed-Multiphor III Electrophoresis System (GE ). I run the gel for 5h, 200V and 15ºC. The genome DNA that Ive loaded were 1 ng/uL, so I visualized with silver staining method. But, after several time Ive conducted this experiment. I can not see my band and I found my band was faint smear. Early, I thought it was caused by the voltage. So, I decrease the voltage to 200V. but, seems it disaster appear again in the same way.

Ive included the image. The left side of lane was the Lambda DNA marker but the phenomenon was similar with the other well. Please…, Please give me some suggestions to solve this problem!! I am desperate!!

sds is used for denaturing page of proteins. you should use tbe or tae for dna.

7.5% is too restrictive for genomic dna. 5% may be better.

on the other hand, most people use agarose to look at genomic dna.

mdfenko on Wed Jul 13 18:20:28 2011 said:

Hi, mdfenko.

The response about my Flatbed is totally awaiting since i posted this. thanks to you.
actually, i was working for optimizing of Flatbed for my IEF project (Multiphor II Electrophoresis system - GE Healthcare). the system doesn't need the liquid buffer as need in ordinary vertical electrophoresis. the buffer used is strip which soaked in Cathode and Anode solution and the Gel was lay onto the ceramic plate which connected with the electricity power.

since i have not generate the result yet, i was still optimizing the convenient way. Actually, if you ever work with this instrument. i am going to appreciate it.. these mess were completely frustrate me.


Thank you..

are you planning to run ief with genomic dna?

ief is for proteins. i'm not sure it would work for nucleic acids.