title: UCSF_MouseArray_1.0_972 [BAC array]
status: Public on Nov 22 2011
contact.city: San Francisco
contact.first: David
contact.last: Quigley
contact.state: CA
contact.address: 1450 Third St. Room 207
contact.department: Helen Diller Comprehensive Cancer Center
contact.country: USA
contact.institute: UCSF
organism: Mus musculus
manufacture_protocol: Isolation of BAC/P1 DNA. We inoculated 0.1ul of a glycerol stock of BAC or P1 containing bacteria into 5 ml LB media containing chloramphenicol (10 ug/ml) or kanamycin (50 ug/ml) for BACs or P1s, respectively and incubated the cultures overnight (~16 h) at 37 degrees C with agitation at 225 rpm. We prepared larger cultures for DNA isolation, by inoculating 25 ml LB media, containing the appropriate antibiotic with 200 ul of the previously grown overnight culture and incubating in a shaking incubator at 37 degrees C at 225 rpm overnight (~16 h). We monitored bacterial growth by measuring the OD of a 1:10 dilution, which preferably ranged between 0.25 and 0.35 at a wavelength of 600 nm. We used the Qiagen Plasmid Mini kit to isolate DNA, following a modified version of the Qiagen Plasmid Mini Purification protocol. We transferred the 25 ml cultures into 50 ml tubes and centrifuged them for 15 min at 4000 rcf at 4 degrees C to pellet the bacteria. We re-suspended the pellet in Qiagen buffer P1 (1.5 ml), containing RNase A at the manufacturer’s recommended concentration and then added buffer P2 (1.5 ml). We mixed the tubes extremely gently by inversion and incubated at room temperature for 5 min. Then, we added buffer P3 (1.5 ml), again mixed very gently by inversion and incubated on ice for 10 min. We inverted each tube once and centrifuged at 4000 rpm at 4 degrees C until the supernatant became clear (45 to 60 min). We filtered the supernatant through a 35-micron nylon mesh prior to loading onto Qiagen-tip 20 columns, which had been equilibrated following the manufacturer’s protocol. We followed the manufacturer’s protocol for the DNA isolation steps with the exception that the elution buffer QF was heated to 65 degrees C before it was added to the column. After elution, we added 0.56 ml isopropanol to the DNA and incubated overnight at 4 degrees C. We collected the DNA pellet by centrifugation for 45 min at 14000 rpm at 4 degrees C. After aspirating the supernatant, we allowed the pellet to dry in air for at most 30 min before re-suspending the DNA in 50 ul of H2O. We determined the DNA concentration using a fluorometer. The yield typically ranged between 60-100 ng/ul (i.e. 3-5 ug per isolation). To ascertain DNA purity, we digested each BAC (200 ng) with HindIII and electrophoresed the digest through a 0.75% agarose gel. We discarded DNA preparations that showed a significant contamination with host bacterial DNA, seen as a background smear of degraded DNA in the gel. Preparation of BAC/P1 DNA representations by ligation-mediated PCR. We digested the DNA with MseI by incubating overnight at 37 degrees C in a 5 ul reaction containing 1.5 ul DNA (20 to 600 ng), 0.4 U/ul MseI (New England Biolabs), and 0.4x One-Phor-All-Buffer-Plus (Amersham). For ligation of adapters, we diluted 1 ul of each digest to a final concentration of 1 ng/ul with H2O and then mixed 1 ng of the digested DNA with 0.5 ul One-Phor-All-Buffer-Plus (10x, Amersham), 0.5 ul of 100 uM Primer 1 (TAACTAGCATGC), 0.5 ul of 100 uM Primer 2 (5’ aminolinker AGTGGGATTCCGCATGCTAGT) and 5.5 ul H2O. We incubated the mixture at 65 degrees C for 1 min after which we ramped the temperature down to 15 degrees C with a ramp-speed of 1.3 degrees C per minute. When the temperature reached 15 degrees C, we added 1 ul of 10 mM ATP and 1 ul T4-DNA ligase (5 U/ul, Gibco BRL) and continued incubation at 15 degrees C overnight. To initiate the first round of PCR amplification, we added a 40 ul mixture, consisting of 3 ul PCR Buffer 1 (Expand Long Template PCR System, Roche), 2 ul of a mixture of each nucleotide (10 mM) and 35 ul H2O to the ligation reaction. Before we started the PCR program, we incubated the reaction at 68 degrees C for 4 min and then added 1 ul DNA polymerase (3.5 U/ul, Expand Long Template PCR System, Roche). We carried out thermal cycling in a Perkin-Elmer Gene Amp PCR System 9700 block as follows: 94 degrees C for 40 s, 57 degrees C for 30 s, 68 degrees C for 1.25 min for 14 cycles, followed by, 94 degrees C for 40 s, 57 degrees C for 30 s, 68 degrees C for 1.75 min for 34 cycles and 94 degrees C for 40 s, 57 degrees C for 30 s and 68 degrees C for 5 min for the final cycle. We electrophoresed 3.5 ul of the PCR product through a 1% agarose gel to determine the size range of the amplified DNA, which ideally ranged from 100 to 2000 bp. To make the DNA for spotting on the arrays, we carried out a second round of amplification in a 100 ul reaction containing 1 ul of the primary PCR product, 4 uM Primer 2, TAQ-buffer II (1x; Perkin Elmer), 0.2 mM dNTP mix, 5.5 mM MgCl2 (Perkin Elmer), 2.5 U Amplitaq Gold (Perkin Elmer) and H2O. We carried out an initial incubation at 95 degrees C for 10 min in a MJ Research Peltier Thermal Cycler 225, followed by 95 degrees C for 30 s, 50 degrees C for 30 s and 72 degrees C for 2 min for 45 cycles and finally 7 min at 72 degrees C. This reaction yields ~10 ug of DNA, with each fragment containing a 5’ amino linker. Preparation of DNA spotting solutions. We evaporated the amplification reaction (100 ul) to a final volume of 50 ul by incubation at 45 degrees C in a hybridization oven (Techne, Hybridizer HB-1D) for approximately 75 min and then added 2.5 volumes of ice-cold ethanol and 0.1 volumes of 3 M NaOAc to precipitate the DNA. (The use of ethanol precipitation proved superior to isopropanol precipitation.) We inverted the tubes and chilled them at –20 degrees C for 15 min before collecting the precipitate by centrifugation at 1699 rcf for 90 min. We washed the pellets with 70% ethanol (150 ul) and collected them by centrifugation at 1699 rcf for 45 min. We air-dried the pellets for approximately 60 to 90 min and then re-suspended them in 12 ul of 20% DMSO in H2O (~0.8 ug/ul). We transferred the DNA solutions into 864 well microtitre plates for robotic arraying. Previously, we prepared DNA for spotting in 80% DMSO and 0.3 ug/ul nitrocellulose. However, we subsequently evaluated spotting solutions containing various concentrations of dimethyl formamide, formamide or DMSO, with or without nitrocellulose. We determined that nitrocellulose was not required for spotting DNA prepared by PCR using primers with 5’amino linkers and that spotting solutions made with 20% DMSO performed well. We have also generated representations of large insert clones for arraying by using degenerate oligonucleotide primed PCR and by amplification of mixtures of subclones from BACs, but found the ligation-mediated PCR procedure to be superior. Array printing. We used a custom built printer, employing a 4 x 4 array of quartz capillary tubes spaced on 3 mm centers to print ~70-100 μm diameter spots on 130 μm centers. We printed each DNA solution in triplicate to create an array of ~7500 elements in a 12 mm square area. We printed the arrays on chromium coated microscope slides (PTI or Nanofilm) for these studies, but also routinely print the arrays on glass slides (Corning GAPs). We allowed the printed slides to dry overnight, then exposed the slides to UV light (65 mJ) in a UV Stratalinker 2400 (Stratagene). We hybridized to these slides without additional treatment, except for the pre-hybridization slide blocking described below. 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.