| Library
Screening Services |
 |
BACPAC Resources has established
a library screening service to assist end-users with identifying
BAC clones from our collection of >100 genomic libraries.
Primary screens are performed via hybridization using synthetic
oligonucleotide probes (an Overgo). The overgo probe is generated
by ordering 24-mers containing an 8 base pair overlap. The
probe is made radioactive in a fill-in reaction containing
32P-dATP, 32P-dCTP (Mc
Pherson et al.). The resulting probe is a highly specific
40mer after denaturation and inclusion in a hybridization
reaction to 22x22cm nylon filters
containing BAC clones spotted in duplicate. Each nylon filter
contains the contents of 48 microtiter dishes X 384 wells/dish
totaling 18,432 clones spotted in duplicate.
Quotes provide end-users
with an estimate for cost associated with a screening project;
all quotes are valid for 30 days. BACPAC Resources assumes
the financial liability for unsuccessful screening projects
consequently end-users are billed on a cost recovery basis.
End-users only pay for successful probes that identify probe-positive
BACs verified in the secondary PCR confirmation assay.
*A limited number of BAC libraries
CHORI-216, CHORI-219, CHORI-303 constructed from the genomes
of Xenopus tropicalis, Xenopus laevis, and Sea Lamprey respectively,
are subject to a non-refundable set-up fee.
| Single
probe library screening |
Price
|
| Small
genomic libraries i.e. invertebrates contained on <4
nylon filters |
$1,500.00
|
| Multiple
probe screening of a small genomic library i.e. invertebrates
(for each additional probe) |
$200.00 |
| 10X
genome equivalents for all BAC library contained on 5-12
nylon filters |
$2,500.00
|
| Multiple
probe screening of a single 10X genome equivalents library
(for each additional probe) |
$400.00
|
| *Non-refundable set-up fee |
$500.00 |
| Library representation or genome equivalents has been
determined using "single copy" gene sequences.
On average 10 probe-positive BACs are identified when
screening a 10X library. Clones 1-10 for each probe are
included in the cost of the screening |
|
| BAC clones >10 are available at prices described
on our website |
|
| Estimated cost for shipping Domestic (USA) |
$35.00 |
| Estimated cost for shipping International depending
on the country |
$55.00 |
Prices are subject
to change without prior notification
| Target
Information / Probe Design / Secondary Screening
|
End-users
provide the following: (1) the library name requested for
screening, (2) an accession number for the target sequence,
(3) and information describing the region within the target
sequence for the probe design e.g.
Library:
RPCI22 Mouse BAC Library
Target
description: myosin, heavy polypeptide
6, cardiac muscle, alpha (Myh6)
Accession:
NM_010856
Description:
target a region 5kb upstream of Exon 4,
and 5kb downstream of Exon 5
DNA sequence within the target region is analyzed using a
probe design script; optimal probes sequences are checked
for target specificity using bioinformatics tools on The UCSC
Genome website (http://genome.ucsc.edu).
Overgos that hybridize to the target sequence, are in conserved
regions of the genome, and don't contain repeat sequences
are optimal probes.
Probe-positive clones
are verified via PCR therefore oligos required for the secondary
screening are ordered in conjunction with oligos for the overgo
probe. PCR primer design is included in the cost for the screening
consequently primers sequences are not required from the end-user.
However if an end-users has PCR primers previous verified
to generate a single band when tested using total genomic
DNA and the primers are within 5-10kb of the target sequence
please include the sequences.
End-users
receive a quote containing relevant information on the library
requested for screening, the gene target and cost for the
screening; a Payment Request Form is also included with the
quote. Once the quote has been accepted and the Payment Request
Form returned the project begins. Required information for
inclusion into the quote:
Name:
Institution:
Mailing
address:
Phone
#:
Fax
#:
Email
address:
| Frequently Asked Questions
|
What
does 10X genome equivalents mean (haploid genome)?
Genome
redundancy is calculated as follows: Clones insert size (kb)
x number of clones / size of the genome (kb); the depth of
the library. A 10X redundant library contains 99% of the DNA
sequences in the genome. On average 10 BAC clones are expected
to contain the target DNA sequence in a 10X library; 5 clones
per haplotype.
How
long does the library screening take after submission of the
completed documents?
The
library screening process takes 4-6 weeks on average for completion.
Laboratory closure during Institutional Holidays can affect
the timelines for project completion.
Modified cloning
vectors for making gene knockout constructs and vectors that
facilitate BAC-end sequencing are actively being pursed in
our laboratory. The BACPAC Resource Center has and will continue
to explore methodologies utilizing BACs for downstream applications.
BACPAC Resource Center is a non-profit organization dedicated
to generating and making recombinant clone library resources
available to the scientific community. Screening high-density
colony filters is an experimental endeavor added to the list
of services intended to aid researcher with the identification
of BAC clones for downstream analysis.
Methodologies
used to screen recombinant clone libraries are robust and
well documented. However some DNA sequences can be problematic
posing difficulties for overgo probe design and colony hybridization.
The BACPAC Resource Center will make a good faith effort to
start and complete all services on time and will notify end-users
if unforeseen problems are likely to cause substantial delays.
The
BACPAC Resource Center does not guarantee that the received
clones are compatible with all down-stream applications.
The
BACPAC Resource Center does not guarantee that the clones
in each well are a homogeneous population. We know that a
limited amount of well-to-well cross-contamination occurs
and is unavoidable when handling arrayed libraries.
The
BACPAC Resource Center reserves the right to discontinue service
for probes and/or problematic DNA sequences. If it is deemed
necessary to discontinue the library screening there will
be no charge to the end-user.
Neither
BACPAC Resource Center nor the end-users shall be responsible
for failure or delay in performance of its obligations related
to the services due to causes beyond its reasonable control,
included but not limited to governmental actions, fire, labor
difficulty, shortages, civil disturbances, transportation
problems, interruptions of power or communications, failure
of suppliers, or natural disasters.
For more information,
please send inquiries to libraryScreening@chori.org
|
