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Resources - Hybridization of High Density Filters

 

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 Resources 
 Hybridization of High Density Filters    

Hybridization of filters can be performed in either rotisserie bottles or heat-seal bags.  In our lab both work well.  We routinely hybridize up to seven filters in one bag and up to five filters in a large (7.5cm diameter) bottle (with nylon mesh between filters).  The filters have been successfully hybridized using a number of different published "Southern" DNA hybridization protocols incorporating a variety of buffer systems.  It has been our experience that all of these established protocols will work with the high density filters.  In our lab we routinely follow the following procedure:

  • Wet filters in minimum volume of  "Church Buffer" in large petri dish.   Keep track of volume of buffer absorbed into the filters as this will contribute to your overall volume.  If using bottles, place nylon mesh between filters as you wet them.

  • Place filters in bottle or bag, add approx. 25ml additional "Church Buffer" and seal.

  • Pre-hybridize one hour at 65.

  • Add labeled probe(s) so that the total cpm of each probe is 1X106 to 11X107.   We routinely label our probes by random prime labeling.  PCR incorporation also works well for smaller (>100-200bp) probes.

  • Hybridize overnight (16-18 hours) at 65o C.

  • Wash filters (in bag or bottle) with Wash I solution at 65o C.  Repeat wash about 4 to 6 times until most of the non-bound probe is removed.  At this point the filters can continue to be washed in the bag or bottle or placed in a large tray in a 65o C water bath for more efficient washing.

  • Wash filters with wash II solution repeatedly at 65o C until the level of cpm (monitored with a Geiger counter) removed remains constant (no longer decreases).

  • Rinse filters in water and wrap individually in plastic wrap.  Take care to remove air bubbles between filter and wrap by pressing with a wipe to force out air.

  • Place two filters in a large cassette (35x43cm), there will be a small overlap of filters but this normally is not a problem in interpreting data.  We place two filters per cassette to maximize film use.

  • Expose for 2 to 72 hours at -80o C.  This depends on the intensity of signal from filters.  We have found it best to develop one piece of film after 4-5 hours exposure to determine the optimum exposure time required for all of the filters.

  • Interpret the position of the positive clones following the protocol supplied with the filters using the transparent overlay as an orientation guide.

  • Store the used filters in a sealed hybridization or zip-lock bag in "Church Buffer" to keep them wet.  Allow the radioactivity to decay out for 2-4 half lives before using again.  We do not recommend stripping the filters as smearing of the DNA may result and the overall life of the filters may also be diminished.

Blocking:
   
We block our nonspecific binding of probes by adding an equal volume of sonicated human placental DNA (10mg/ml) to our probe solution, incubate one hour at 65o C before adding the probe to the hybridization bag/bottle.  Alternatively, the sonicated human placental DNA can be added during the pre-hybridization to the bag/bottle at a level of 100-200 ug/ml of hybridization solution.

Solutions:

Church Buffer -- 1 Liter BSA 10 gm
  0.5M EDTA 2 ml
  1 M NaHPO4(pH: 7.2) 500 ml
  20% SDS 350 ML
  H20 to 1 liter

Wash I -- 2 Liters BSA 10 gm
  0.5M EDTA 2 ml
  1 M NaHPO4(pH: 7.2) 80 ml
  20% SDS 500 ml
  H20 to 2 liters

Wash II -- 4 liters 0.5M EDTA 8 ml
  1 M NaHPO4(pH: 7.2) 160 ml
  20 % SDS 200 ml
  H20 to 4 liters

1 M NaHPO4(pH: 7.2) -- 1 liter Na2HPO4-7H2O 134 gm
  85% H3PO4 4 ml

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