Hi,
can you describe me how an hybridization oven works for southern blotting and where/how I put the membrane in it? I need to do a southern but it is the first time and I am not supervised , so it is difficult to figure out. How do I use the hybridization bags?
Thanks in advance
These bags are messy, especially if you use radiolabels like 32-P. A hybridization oven uses roller bottles, which are easier and cleaner to use. Bags and roller bottles were developed to keep the hybridization volume small, 10ml or less, because the probe is limited, small volumes are essential to drive the kinetics of hybridization. Hybridization temperatures are usually about 65oC in a salt buffer. If using formamide, use lower temp. Much of this is explained in the Maniatis book.
Place dried membrane into the bottle. Add a pre-hybridization solution to wet the membrane. Include denatured salmon sperm DNA as a blocking material (0.1 mg/ml). After 1 hour or so, add the heat denatured probe. Use about 0.5-1.0 E6 counts per ml if you are using a 32-P labeled DNA fragment. The fragment must be labelled to high specific activity 1E8 cpm per ug or so. Hybridize overnight, then wash 2 times with 2XSSC, 1% SDS, then 2x with 0.2X SSC, 0.1% SDS at 65oC for 30 minutes each time. If using a geiger counter, the bot should have very low signal, just a few counts above background. Expose to film overnight at -80oC.
I like bags although if you get bubbles of air trapped inside them you can get some edge effects where the perimeter of the bubbles can leave a Mark on the membrane. Bottles are good but 2 additional things from Gregory!so excellent answer. If the membrane is large then use a plastic mesh to stop the membrane from touching itself when you get poor hybridisation and washing so some dirty results where ther is overlap. Also be careful with the direction that you wind the membrane compared with the direction of rotation if you are using a rotating hybridisation oven or the membrane will roll up small and not hybridise well. If this happens just turn the bottle round and the membrane will unroll again
Thank you. So if I used bags, I should remove air from inside, shouldn't I? Do I need to create vacuum?Sorry for the question, I have no idea of how they work
Remove as much air as possible then seal the bag. I used lay flat tubing and a het sealer and on pre hybe seal with about 3cm spare so that you can cut the bag at the end, add the probe and then re seal. Don't worry too much about bubbles but it is best not to have froth so handle the bag gently and ideally have it moving gently like on a rocker while pre hybe and hybridising to ensure that all area of the filter are exposed to all solutions equally. No vacuum just squeeze out most of he air before sealing. Also as Gregory says do not use too much volume of hybridisation solution as the higher concentration of probe means that it reaches full hybridisation quicker in a small volume
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