PROTOCOL

Hybridization of Clemson High-Density BAC Filters

(07/08/02, Varma Penmetsa: rvpenmetsa@ucdavis.edu)

Pre-hyb 1 membrane / bottle, wrapped in nylon, print side facing in (= colony side facing inwards).

Pre-hyb 16-24 hrs @ 65C with 50ml solution per bottle (0.25M NaP pH 7.2 buffer; 7% SDS, 1mM NaEDTA).

Hybridization:
Discard pre-hyb buffer.
Add 20 ml/bottle fresh pre-heated hyb buffer.

Add label (2 std lab scale rxn /probe for 2 bottles i.e, 1 rxn of probe/bottle). Reset temp for hyb to 60oC. Hyb 14-18h at ~3 rpm.

Washing:
Discard hyb solution into radioactive liq waste. Rinse bottle briefly with preheated (60oC) first wash solution (2X SSPE; 0.25%SDS). Rinse in bottle in hyb oven twice 30 min. each in 75 ml/ bottle of 2X SSPE; 0.25%SDS.

Transfer membranes to 1 gallon tupperware plastic box. Rinse in 500ml of 1X SSPE; 0.1% SDS @ 65oC for 20 min on shaker-waterbath

Rinse once @ 65oC for 20 min on shaker-waterbath in 500ml of 0.5X SSPE; 0.1% SDS.

Dry membrane briefly on 3mm filter-paper to remove dripping rinse buffer.

Place membrane in Clingwrap (preferably, and fold over so membrane is covered on both sides. Saranwrap may be OK, but it is thicker and more brittle tearing easily.

Transfer to auto-radiograph ("X-ray" film), backed with an intensifier screen and store at -80oC. Typically, for homologous probes (Mt sequence), an over night exposure provides sufficient signal. Using 2 films and screens will allow for checking if longer exposures are required. Alternatively, use the additional film for a longer exposure to bring out background on the membranes which greatly helps in orienting the membrane for clone ID.

REUSE OF MEMBRANES:
To best extend the useable life of the membranes, it is best to avoid the standard probe stripping methods (with NaOH or boiling SDS). We do the second probing on an un-stripped membrane and ignore the positives from earlier rounds of hybridizations. We perform stripping of membranes only if the probe used lights up highly repeated sequences that give several hundred positives. The advantage of overlay hybridization (without an inter-veining stripping is that the positives from the first probe actually help in aligning the membrane for reading out positives from the subsequent probe(s)).