BAC-filter hybridization
From Wikionchus
BAC filter hybridizations are methodically very similar to Southern Blots. A radioactively labelled probe is hybridized to filters that are spotted with several thousand BAC clones. BAC DNA is spotted at characteristic positions. Each BAC is spotted twice to unambiguously score positives. The filters are organized in 48x48 squares; each square (depending on the library) contains a certain number of spottable positions. The exact arrangement differs between the HindII and EcoRI library of Pristionchus pacificus.
Contents |
A BAC hybridization consists of 4 steps
- Generating the probe
- Hybridization
- Washing and exposing BAC filters
- Correct storage of decaying and cold BAC filters
Generating the probe
Usually PCR products of a minimum size of 100 bp are used as probes. In practice we only use Klenov-labelling with random hexamer oligos. We use the MEGAPRIME random labelling kit (Amersham Biosciences). The obvious advantage over PNK/end labelling are the high specific activity and therefore the short exposure times needed to visualize a signal. Before working with radioactivity get an introduction (Werner Mayer).
- Gel-purify the PCR product.
- Use 30-50 ng of DNA for labelling.
- In order to minimize exposure to radioactive compounds prepare all master mixes oi the cold-labs and only then go to the hot-lab.
- In a Eppendorf tube mix
- 30-50 ng DNA
- 4 µl 5X labelling buffer (contains all dNTPs except for dCTP)
- 2 µl random primer solution
- H20 to 18 µl
- Denature at 95°C for 5 minutes; place on ice immediately to avoid renaturation.
- Add 1µl of alpha-P32-dCTP
- Add 1µl of Klenov enzyme (you need 1 U).
- Pulse spin and incubate at 37°C for 30 minutes.
- Before starting the labelling reaction prehybridize BAC filters in BAC hybridization buffer at 42°C.
Hybridization
- Two BAC filters can be hybridized in the same roller bottle; make sure to separate them with the plastic meshes before rolling them up.
- Prepare a positive control by spotting 0.2 µl of the gel eluted PCR product on a small piece of Hybond N membrane, denature DNA by dropping 9 µl of 0.1 M NaOH on the DNA. Crosslink in the UV-crosslinker with the "Auto crosslink" setting.
- Prehybridize BAC filters in 30 ml BAC hybridization buffer at 42°C.
- Make sure that the roller bottles are tightly closed.
- Denature the labelling reaction mix by adding 94 µl H20 and 6µl NaOH 2M.
- Split total volume (120 µl)into two aliquots. Only one is needed per roller bottle.
- Add 60µl to the prehybridized BAC filter/s.
- Hybridize at 42°C with constant rolling for at least 10 hours.
Washing and exposing BAC filters
- Remove the hybridization solution by carefully pouring it into the radioactive waste containers. Make sure to use the ones for P32!
- Add 30 mls of 2 X SSC, 0.1 %SDS.
- With constant rolling, heat eat the incubation oven to 62°C.
- Wash for 30 minutes at 62°C.
- Exchange the first washing solution with preheated 0.2 X SSC, 0.1 % SDS.
- Wash for another 30 minutes.
- Carefully drain second washing solution into waste containers.
- Take out BAC filters and positive controls ans heat-seal them in plastic bags.
- Tape them into an exposure cassette.
- The x-ray films are in the cold-room on the top-shelf to the left.
- Go to the dark-room and make sure to lock the door.
- Place x-ray film onto the BAC filter and close exposure casette thightly.
- Place cassette into -80°C freezer for 6-20 hours. Depending on the speciifc activity (means: age) of the gamma-P32-dCTP it may take up to 2 days before signals can be seen.
- One potential problem is that although one can see signals the background is so low that the grid can not be seen. In that case: congratulate yourself for working so clean :), expose longer and hope for more background.
- Before using the developing machine make sure that there is enough fresh developing and fixing solution in the storage containers.
Correct storage of decaying and cold BAC filters
Links
- Back to Pristionchus pacificus Protocols page
