Southern blotting problem (no ladder, high background) - (Nov/15/2015 )

This is my Southern blot using 32-dCTP labeling.

There are two problems

One is that there’s no ladder. (first lane and last lane)

The other is the huge weird background. (very clear black part in the upper and white in the bottom part)

Here are the details of my experimental procedure:

I tried to genotyped the recombinant yeast strains, so I treat the genomic DNA with XbalI for 37C 4hr. Then I loaded my samples on the 0.8% agarose gel and run at 60volt O/N.

Then I check the gel and found that the DNA was cut appropriately.

After that I start the Southern electro-transferring for 2 hr. Before the hybridization, I soaked the membrane with 0.8N NaOH (250ml) for 15 min and then change the sol to 10X SSC buffer for another 15 min.

Next, I washed the membrane with 0.5M phosphate buffer in the Southern glass tube. And then I pour 25ml Southern hybridization buffer (65C) into the tube and put it in the oven (65C) for 1 hr.

At the same time I prepared my mixed probes which contain: 100ng of my PCR probe, 0.1ng lambda DNA BstEii and some MQ water.

First I keep the mixed probes at 100C for 3 min and then put it on ice.

Next, add 5ul High primer mix and 4ul 32P-dCTP. Incubate the mixture at 37C for 20min.

Add the mixture into a Mini Quick Spin DNA column, and centrifuge the mixture at 3400rpm for 4 min.

Check the radioactivity of the mixture using gager counter

Incubate the mixture at 100C for 3 min, then put it on ice

Add the mixture in hybridization buffer and pour it in the glass tube (with membrane inside)

Rotate the glass tube at 65C overnight

Wash the membrane four times (15min each with wash buffer at 65C)

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Sorry about the lengthy experiment procedure typed above…

Any comment is welcome!!

Peggy

the background is not a significant issue. it looks like the buffer front is the border between light and dark. it does look like uneven blocking.

why would you expect the ladder to show? is it radioactive with 32p?

I don't understand about the buffer front...actually I roll the membrane from the long- edge and put it into the glass hybridization tube. 

I also use the same hybridization buffer for Pulse-field gel but it did not show the black white border.

Also, my mixed probes contain: 100ng of my PCR probe, 0.1ng lambda DNA BstEii (the ladder), so presumably after P32-dCTP labelling my ladder on the membrane should be hybridized and show signal too...

the buffer front is from caused by migration of buffer components through the gel. if you run the gel further then you should be able to eliminate the front if it bothers you (you may have a tracking dye running with the front, if so then just run it out of the gel to ensure you don't see the front).

the uneven background indicates that you are not evenly spreading the prehyb or hybridization solution (or both).

you may want to try phosphorylating the ladder separately from the probe and then confirming proper incorporation before mixing with the phosphorylated pcr probe or just run the radioactive ladder on the gel.