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Platform GPL4714 Query DataSets for GPL4714
Status Public on Jan 04, 2007
Title RPCI Mouse 6.5K
Technology type spotted DNA/cDNA
Distribution non-commercial
Organism Mus musculus
Manufacturer RPCI Microarray and Genomics Facility
Manufacture protocol Isolation of BAC plasmid DNA: BAC clone plasmid DNAs, used as templates for PCR representations are prepared using a Qiagen BioRobot 3000. Each clone is retrieved from archival copies of the RPCI-23 BAC Resource, maintained at RPCI. The clones are streaked to single colony, grown overnight (16-18 hours) as 1.0ml pre-cultures in 96 deep-well plates at 37 C in a New Brunswick environmental shaker, and then re-inoculated as 2ml prep-cultures in 48 deep-well plates. After incubation, the cultures are RNase treated and prepped in the BioRobot 3000 using R.E.A.L. Prep 96 BioRobot kits (Qiagen). The BAC DNAs are resuspended in TE and serve as templates for restriction digestion and PCR amplification. We have found that Qiagen prepared DNA is of high quality and outperforms phenol:chloroform extraction methods for the highly sensitive restriction digestion and ligation steps.
Ligation of adapters: The adapters are ligated to the digested DNA by first annealing Mse-21 and Mse-12 primers. 1 ul of each digest (1 ng/ul), is added to 0.5 ul of 100 uM Mse-12 (TAACTAGCATGC), 0.5 ul of 100 uM Mse-21 (5’ aminolinker AGTGGGATTCCGCATGCTAGT) in a final volume of 7.5 ul using a thermalcycler (MJ Research). The mixture is incubated at 65?C for 1 min followed by ramping the temperature down to 15 C with a ramp-speed of 1.3 C per minute. When the temperature reaches 15 C, ligation proceeds with 1 ul of 10 mM ATP, 0.5 ul One-Phor-All-Buffer-Plus (10x) and 1 ul T4-DNA ligase (5 U/ul, Invitrogen) added to each tube and incubated at 15 C overnight.
PCR amplification: Two rounds of amplification are required to produce enough product for array generation as described in Cowell, J. K. and N. J. Nowak. 2003. High-resolution analysis of genetic events in cancer cells using bacterial artificial chromosome arrays and comparative genome hybridization. Adv Cancer Res 90:91-125. A single BAC PCR-representation produces enough printing solution for over 4,000 arrays. Preparation of DNA spotting solutions: PCR products (100 ul) are purified for printing by a series of ethanol precipitation steps, followed by resuspension of the pellets in 20 ul of 25% DMSO in H2O (final DNA concentration ~0.8 ug/ul) using a Qfill2 (Genetix). The DNA solutions are rearrayed into Genetix 384-well V-bottom plates using a Hydra 96-PP liquid handling system, and stored at 4 C until printing. We determined that DNA prepared using LM-PCR performed superiorly over other methods, including degenerate oligonucleotide primer PCR, inter Alu PCR and amplification of BAC subclones mixtures. 25% DMSO has several properties that make it an ideal solution for resuspending the amplicons. First, it acts as a denaturant, rendering the printing solution single-stranded for optimal slide binding and subsequent hybridization. Secondly, DMSO does not evaporate, so the printing solution volume is not affected by exposure to the environment during multiple print runs.
Array printing: Slides are printed using a MicroGrid ll TAS arrayer (Genomic Solutions) using 10K Microspot pins at 48% relative humidity, 22?C. We have a climate controlled, HEPA filtered room to minimize environmental changes that can affect printing. In addition, the arrayer is supplied with HEPA filtered air to further maintain a dust-free environment. We print each BAC clone in triplicate to create an array of on amino-silanated glass slides (Schott Nexterion, Type A+). The printed slides dry overnight, and are UV-crosslinked (350 mJ) in a Stratalinker 2400 (Stratagene). The slides are visually inspected and stored in a dessicated environment and are hybridized without additional treatment. Attempts at post-print processing or inclusion of pre-hybridization steps resulted in background issues that made the images difficult to analyze. We found no indication of DNA loss from the spots at any stage of the procedure when we performed hybridization in formamide buffers at 37°C (via DAPI staining).
Web link
Submission date Jan 02, 2007
Last update date Jan 05, 2011
Contact name Devin McQuaid
Phone 716-845-8805
Organization name Roswell Park Cancer Institute
Lab Microarray and Genomics Facility
Street address Elm and Carlton
City Buffalo
State/province NY
ZIP/Postal code 14263
Country USA
Samples (99) GSM154569, GSM154570, GSM154571, GSM154572, GSM154574, GSM154616 
Series (3)
GSE6706 A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells
GSE22927 Hierarchical synergy of Pten, p53 and Rb pathways in high-grade astrocytoma induced in adult brain
GSE40106 Pten deletion in neonatal brain induces an abnormal neural progenitor niche that can synergize with Trp53 loss to generate medulloblastoma

Data table header descriptions
BAND Band Clone is in
Chr Chromosome
Start Start location in base pairs
Stop Stop location in Base pairs
Center Center location in base pairs

Data table
ID BAND Chr Start Stop Center CLONE_ID
RP23-271O17 qA2 chr1 3009947 3236708 3123327.5 RP23-271O17
RP23-232J11 qA2 chr1 3501969 3677784 3589876.5 RP23-232J11
RP23-320L20 qA2 chr1 3983353 4186213 4084783 RP23-320L20
RP23-445L18 qA2 chr1 4431263 4611118 4521190.5 RP23-445L18
RP23-131C23 qA2 chr1 4848346 5074306 4961326 RP23-131C23
RP23-32F6 qA2 chr1 5322154 5552240 5437197 RP23-32F6
RP23-297K4 qA2 chr1 5817753 5998723 5908238 RP23-297K4
RP23-422K10 qA2 chr1 6286858 6484497 6385677.5 RP23-422K10
RP23-69J22 qA2 chr1 6786571 7005553 6896062 RP23-69J22
RP23-294B12 qA2 chr1 7320262 7495777 7408019.5 RP23-294B12
RP23-410H10 qA2 chr1 7804987 8011264 7908125.5 RP23-410H10
RP23-315F19 qA2 chr1 8281950 8444731 8363340.5 RP23-315F19
RP23-144B15 qA2 chr1 8768706 8986409 8877557.5 RP23-144B15
RP23-480F21 qA2 chr1 9248048 9412051 9330049.5 RP23-480F21
RP23-280K9 qA2 chr1 9649054 9861794 9755424 RP23-280K9
RP23-291G6 qA2 chr1 10114300 10330740 10222520 RP23-291G6
RP23-272K15 qA3 chr1 10606416 10822744 10714580 RP23-272K15
RP23-303L3 qA3 chr1 11022875 11230546 11126710.5 RP23-303L3
RP23-50B16 qA3 chr1 11496204 11852802 11674503 RP23-50B16
RP23-32B12 qA3 chr1 12023731 12243241 12133486 RP23-32B12

Total number of rows: 6507

Table truncated, full table size 387 Kbytes.

Download family Format
SOFT formatted family file(s) SOFTHelp
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Supplementary data files not provided

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