(Protocol for a single high-density BAC colony filter (HDR filter) of 22 cmx22 cm) to be screened using Amersham''s ECL hybridization kit; this protocol was kindly contributed by Dr. Kazuei Mita from the National Institute of Agrobiological Science, Owashi 1-2, Tsukuba, Ibaraki 305-8634 , JAPAN)
Amersham Biosciences Catalog #RPN3001
Take 500 ng DNA and 0.25 ng pBR322 in a Eppendorf tube, and adjust the volume to 16 ul by dH2O. (In our case, 10 ul d H2O + 1 ul pBR322 + 5 ul PCR product)
Heat at 95C for 5 min, then immediately cool on ice for 3 min.
Spin down briefly.
Add equal volume of DNA labeling reagent (16 ul), and mix gently.
Add glutaraldehyde solution equal volume (16 ul) to the labeling reagent, mix thoroughly.
Incubate for 10 min at 37C.
(The probe can be held on ice for 10-15 min.)
Take 25 ml hybridization buffer in a beaker.
Add 0.86 g NaCl to make 0.5 M, and add 1.25 g blocking agent to final concentration of 5%.
Mix and stir for more than 15 min at room temp.
Then, heat to 42C.
(Use 20 ml for pre-hybridization and 5 ml for probe solution.)
Pre-hybridization of filters: more than 30 min at 42C
After pre-hybridization, transfer the hybridization solution to 50 ml tube, add labeled probe (48 ml), mix well and put the hybridization solution back to HDR filter for hybridization.
Hybridization : Overnight at 42C
While hybridizing, prepare the primary washing solutions, 0.5x SSC/0.4% SDS and 0.1x SSC/0.4% SDS , and pre-warm these at 55C.
Washing of Filter(s):
Wash HDR with 150 ml 0.5x SSC/0.4% SDS for 10 min at 55C.
Wash HDR with 150 ml 0.1x SSC/0.4% SDS for 10 min at 55C.
Then, wash with 500 ml 2x SSC for 5 min at room temp., and repeat one more time.
Detection:
Mix 7.5 ml detection reagent 1 and 7.5 ml detection reagent 2. (We normally use this amount for four filters.)
Wet a paper towel on a flat container (N.B. remove bubbles trapped).
Place the filter on the paper towel wetted with detection reagents, DNAside up.
Warning : Never let the filter dry up.
Incubate for 1 min at room temp.
Drain off excess detection reagents and wrap with Saran Wrap.(Remove air).
Autoradiograph
Transfer the filter ( DNA side up) into the cassette.
Place an X-ray film in the dark on top of the filter and mark the filter number with a pen in a corner.
Exposure time: 30 min. (This is not the exact time. The optimum exposure time strongly depends on the conditions of the individual experiment)
Notes:
In Dr. Mita''s laboratory, the hybridizations are done in a hybridization oven (Amersham) with 6 glass tubes (4cm in diameter; 24 cm in length).
The probe DNA length should be more than 400 bp. If the DNA is shorter than 400 bp, the signal becomes too weak to detect.
Disclaimer added by BACPAC Resources Center (BPRC) staff:
These procedures have worked very well for a large number of filters generated for the Silkworm BAC and fosmid libraries. However, high density colony BAC filters for other libraries have not, and will not, be tested for compatibility with this protocol. When ordering filters to be used for non-radio-active hybridizations, BPRC will not guarantee that the filters will be compatible with this procedure. Most likely, however, they will be compatible with this protocol.
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