384 Colony DNA Membrane
Contents and Storage
Shipping and
Storage
The 384 Colony membrane is shipped at room temperature. Upon receipt, store
the membrane at room temperature.
Types of Products
This manual is included with the following products. For more details on each
clone collection, see next page.
Product
Catalog no.
CITB Human B & C BAC 384 Colony Membrane
96056
CITB Mouse BAC 384 Colony Membrane
96051
Contents
Each 384 Colony Membrane contains 384 clones from a BAC library arrayed on a
nylon membrane. The BAC library is constructed in pBeloBAC11 vector and
transformed into GeneHogs® (HS996) E.coli. The membrane is supplied enclosed
between 2 filter paper sheets.
Quality Control
Each BAC library is qualified by growth in LB medium containing 12.5 µg/ml
chloramphenicol at 37°C overnight. The libraries exhibiting growth under these
conditions are spotted on the membrane.
25-0642 Version B
March 31, 2003
Using 384 Colony Membranes
Introduction
The 384 Colony Membranes are ideal for identifying clone address by
hybridization. Information on the clone collection and membrane production is
included in this section.
Clone Collection
Overview
The table below provides an overview of the different BAC Clone Collections,
source, coverage, and vector information.
BAC Clone
Collection
Source
Vector
Average
Insert Size
Host
Coverage
CITB Human
B&C
978SK cell line
(1-194, 384-well plates)
Human sperm
(195-768, 384-well plates)
pBeloBAC11
130 kb
GeneHogs®
(HS996)
15x
CITB Mouse
CJ7 ES cell line/129Sv
(1-612, 384-well plates)
pBeloBAC11
130 kb
GeneHogs®
(HS996)
8x
CITB
The CITB BAC Libraries were constructed through collaboration with Hiroaki
Shizuya at the California Institute of Technology.
The DNA source used for constructing the library is listed in the above table. The
CITB Human B & C and CITB Mouse libraries were constructed by cloning the
DNA into the Hind III sites of pBeloBAC11 vector and transformed into
GeneHogs® (HS996) E.coli. The library is arrayed into 384-well plates. For details
on library construction, visit http://www.tree.caltech.edu/
Membrane
Production
The 384 Colony DNA membranes are manufactured by spotting BAC library
clones directly from a 384-well plate onto a nylon membrane. The BAC colonies
from each plate are grown on the membranes and then processed according to
standard alkaline lysis procedures. The DNA is cross-linked to the membrane
using ultraviolet light.
Each membrane contains colonies from a single 384-well microtiter plate. The
plate identity is noted on the DNA side of the membrane and plate number is
indicated on the A1 corner of the membrane.
Vector Information The BAC library is constructed in pBeloBAC11 vector. For vector map and
cloning site, see page 4. The complete sequence is available on our Web site
(www.invitrogen.com).
Continued on next page
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Using 384 Colony Membranes, Continued
Hybridization
Guidelines
Hybridization
Conditions
•
You may use purified DNA inserts, BAC and PAC insert DNA, or
oligonucleotides (of more than 40 bases) to prepare probes
•
Use probes with specific activity > 5 x 108 counts per minute (cpm)/µg to
obtain the best results
•
Avoid touching the membrane with bare hands
•
Always wear gloves and lab coats
•
Maintain a hybridization volume of 25-30 ml per membrane to prevent the
membranes from folding or adhering to each other
You may use any hybridization method of choice. Hybridizations are usually
performed using a standard hybridization buffer containing SSC, SDS,
Denhardt’s reagent, salmon sperm DNA, and formamide at 42 °C for 16 hours.
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Map and Cloning Site of pBeloBAC11
Map of
pBeloBAC11
The figure below shows the features of pBeloBAC11 vector. The complete
sequence of pBeloBAC11 is available for downloading from our Web site
(www.invitrogen.com) or by contacting Technical Service (see page 6).
Sal 1
loxP
cosN
Not l
lac Z
Not l
rC
pa
par
B
r
CM
pBeloBAC 11
7.4 kb
par
A
S
i
or
r ep E
Cloning Site of
pBeloBAC11
The cloning site for pBeloBAC11 is shown below. Restriction sites are labeled to
indicate the cleavage site. The inserts are cloned into the Hind III site.
T7 Promoter Primer
EcoR I
266 AGGGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GAATTGTAAT ACGACTCACT ATAGGGCGAA
Kpn I Sma I BamH I
Xba I
Sal I
Pst I
Sph I Hind III
Sp6 Promoter Primer
336 TTCGAGCTCG GTACCCGGGG ATCCTCTAGA GTCGACCTGC AGGCATGCAA GCTTGAGTAT TCTATAGTGT
406 CACCTAAATA GCTTGGCGTA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACATTCC
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Accessory Products
Additional
Products
Additional products that may be used for screening and hybridization of the
384 Colony Membrane are available from Invitrogen. Ordering information is
provided below.
Item
Quantity
Catalog no.
100 ml
750018
UltraPure Salmon Sperm DNA
5 ml (5 x 1 ml)
15632-011
UltraPure™ 20X SSC
1L
15557-044
500 g
15515-026
50X Denhardt’s Solution
™
™
UltraPure Formamide
Individual Clones
Individual clones are available as glycerol stocks for purchase after the
membrane is screened. The individual clone is identified by the row and
column number of the plate spotted on the membrane.
DNA Labeling
Systems
A variety of DNA Labeling Systems are available from Invitrogen to enable
synthesis of radioactively labeled probes for hybridization protocols. For more
information, contact Technical Service (see next page) or visit our Web site
(www.invitrogen.com).
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Technical Service
World Wide Web
Visit the Invitrogen Web Resource using your World Wide Web browser. At the site, you can:
•
Get the scoop on our hot new products and special product offers
•
View and download vector maps and sequences
•
Download manuals in Adobe® Acrobat® (PDF) format
•
Explore our catalog with full color graphics
•
Obtain citations for Invitrogen products
•
Request catalog and product literature
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http://www.invitrogen.com
...and the program will connect directly. Click on underlined text or outlined graphics to
explore. Don't forget to put a bookmark at our site for easy reference!
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To request an MSDS, visit our Web site at www.invitrogen.com. On the home page, go to
‘Technical Resources’, select ‘MSDS’, and follow instructions on the page.
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For research use only. Not intended for any animal or human therapeutic or diagnostic use.
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