| Screening
BAC filters with non-radioactive probes |
 |
(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)
Probe
labeling (
ECL
Direct Nucleic Acid Labeling & Detection System.
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.)
Hybridization
buffer
- 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,
DNA
side 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.
References:
Koike
Y., Mita K., Suzuki M. G., Maeda S., Abe H., Osoegawa K.,
deJong P. J., Shimada T. Genomic sequence of a 320-kb segment
of the Z chromosome of Bombyx mori containing a kettin ortholog.
Mol Gen Genomics 269:137-149 (2003)
Web
page updates: August 25, 2005;
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