Got problems about Southern blotting...
There are two problems for my Southern blot using 32-dCTP labeling.
One is that there’s no ladder.
The other is the huge weird background. (very clear black in the upper part and white in bottom part)
Here is 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. [Pre-hybridization]
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)
Expose the membrane.
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Sorry about the lengthy experiment procedure typed above…
Any comment is welcome!!
Peggy
If you either use (P)32-dCTP nucleotides for PCR or labeled primers, only your amplified DNA will appear in your Southern blot, since the ladder is not labeled.
There are some protocols that you can use for labeling your ladder:
The picture shows a background similar when the membrane gets dry, which increases background. Check for your hybridization protocol to make sure this do not happen.
Hi sir!
- For the background problem) I believe that the membrane is not dry after the 0.5M phosphate buffer wash since it stayed inside the glass tube (with either hybridization buffer or wash buffer) until I expose it.
- For the ladder problem) I prepared my mixed probes which contain: 100ng of my PCR probe, 0.1ng lambda DNA BstEii (the ladder!). After 100C 3min, the dsDNA will be denatured. Then I add the primer mix and P32-dCTP into the mixture for labelling reaction, so the newly synthesized daughter strands of my PCR probe and ladder should be P32 labelled...
- https://www.neb.com/products/n3014-dna-bsteii-digest
Thanks!
My pleasure.
If you have access to a real time thermal cycler with HRM capabilities, you can also do high resolution melting analysis (HRM), which has advantages for discovering new SNPs, although it does not work in all SNPs.
Check this paper (sorry I do not have any published of my own):
http://www.sciencedirect.com/science/article/pii/S0009898111001549
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