title: Compugen mouse 22K array (UQ)
status: Public on Feb 27 2008
contact.city: Melbourne
contact.first: Yi Mo
contact.last: Deng
contact.email: dengym@unimelb.edu.au
contact.state: Victoria
contact.address: Royal parade
contact.department: Medicine
contact.country: Australia
contact.institute: University of Melbourne
organism: Mus musculus
manufacture_protocol: POLY-LYSINE SLIDE PREPARATION:
  This protocol follows the Stanford protocol (Pat Brown Lab at Stanford), except for the exception of glass source and then number of slides processed (max is 20 Vs 60 due to slide racks we use and spinning restrictions). We have tried a variety of glass types and found Menzel& Glaser suprefrost plus to be a great substrate.
  Background problems: We have made an awful lot of crap batches of poly-lysine slides. Common causes of bad batches include using dirty glassware or forceps, using cheap ethanol, using slide racks not dedicated to this process and having talc run off wet gloves during the rinsing steps and onto the slides.
  Plunging: One of the major factors in getting a uniform coating on slides is the thoroughness of the slide agitation when moved between different solutions. It is essential that slides be plunged in and out to ensure no gradient of mixed solution is left between the slides. We have found that "jiggling" the slides in the rack ensures an even coating, even to the edge of the slide that is resting in the rack. Getting the knack of this can be tricky. Excessive jiggling leads to all the slides coming out of the rack..
  Reagents Qty
  Slides  2 pkts Menzel & Glasser Superfrost PlusTM microscope slides.
  MilliQ H2O  760mL
  NaOH  190 g
  95% Ethanol  1140 mL
  Poly-L-lysine  70 mL Sigma #P 8920
  PBS  70 mL
  Equipment
  Oven (45oC)  1
  Slide rack  4 Kartel polypropylene slide rack with handle (holds 20 slides). We Shorten the handles down to approximately 5cm long so that the rack can be directly spun in a bench top centrifuge with microtitre plate buckets.
  2L Beakers  4 2L beakers should dedicated to slide preparation.
  Slide storage box  1 (holds 100 slides).
  Stirring bar  2
  Stirring unit  2
  Stirring bar cover  2 Home made arch that goes over the stirring bar to allow mixing. while washing and coating slides.
  Flat nose Forceps  1 Used for loading slides into racks.
  Large forceps  2 Used for handling slide racks
  2L measuring cylinders  2 Polypropylene.
  POLY-LYSINE SLIDE PREPARATION:
  Place 80 slides into 4 slide racks using forceps.
  Prepare cleaning solution as follows: Dissolve 190 g NaOH in 760 mL H2O. Add 1140 mL of analytical grade ethanol. Place a stirring bar in the bottom of the beaker and mix until completely dissolved. CAUTION, THIS SOLUTION IS EXTREMELY CAUSTIC.
  Place a "stand" over the stirring bar. We use a polypropylene lid of a p200 micropipette tip box with the ends cut off as an arch over the stirring bar.
  Submerge slides in their racks 2-3 times to ensure even wetting. Jiggle in the rack just enough to have the slides momentarily lose contact with the rack. Stack on top of the arch and stir as quickly as possible for 2 hr.
  Fill 2L beakers with ddH20. Handle the slide racks with clean forceps and quickly transfer one rack at a time to 1.8L of ddH2O. Rinse vigorously by plunging racks up and down. Plunging should once again jiggle the slides so they lose contact with the sides of the rack.
  Repeat rinses 5X with fresh ddH2O each time. It is critical to remove all traces of NaOH-ethanol. Once slides are clean, they should be exposed to air as little as possible. Dust particles will interfere with coating and printing.
  Prepare Poly-lysine solution as follows: 200 mL poly-L-lysine + 200 mL tissue culture PBS in 1600 mL water. Use plastic graduated cylinder and beaker.
  Transfer slides to circulating poly-lysine solution. Jiggle 5 times, and shake for 1 hr.
  Transfer rack to fresh chambers filled with ddH2O. Plunge up and down 5X to rinse.
  Centrifuge slides on micro-titer plate carriers (place paper towels below rack to absorb liquid) for 5 min. @ 500 rpm. Transfer slide racks to plastic slide chambers with covers.
  Dry slide racks in 450C vacuum oven for 10 min. (Vacuum is optional).
  Store slides in closed slide box (without rubber or cork mat in the bottom).
  ELEMENT PREPARATION:
  Elements are normally prepared from plasmid DNA or cleared bacterial lysates. The percentage of PCR failure for both methods is typically 5-7%. Plasmid DNA templates were also for sequencing of elements for QC purposes.
  Cleared bacterial lysates: from Biotechniques, 29(3), Sept 2000,548-562
  Selected clones are inoculated into a 96 well, U bottom plate containing 100 µl of LB/10% glycerol/Ampicillin (50µg/ml) using a biomek 2000 HDRT or 5µl of liquid culture. The plate is sealed and incubated o/n at 37ºC.
  5µl of culture suspension are transferred into a 96 well U-bottom plate containing 95µl of MilliQ water and heated to 95oC for 10 minutes in a laboratory oven to lyse the cells and release the plasmid clones. Caution: some ploy carbonate plates warp at this temperature.
  Precipitate cellular debris by centrifugation at 1500´g for 5 minutes
  Take 5µl of lysate as template for PCR. Store the remainder frozen. Re-spin before further use.
  Plasmid preparation:
  This protocol is a modified version of the Beckman plasmid preparation procedure for the Biomek 200 but we use multi-channel pipettes instead. Protocol yields small amounts of DNA for a mini prep but the DNA is good enough to be sequenced if coming from the IMAGE bug strain.
  Aliquot Inoculate 1.1ml of LB broth + ampicillin 50µg/ml into 2.2ml deep 96 well plates ABgene. Square well, round bottom plates are best as they are easier to remove supernatant from. Cover plate with sealable lid and shake at 250rpm (minimum) overnight at 37oC.
  Spin down bacterial pellet by spinning plate at 3K for 15min 4oC. Tip off supernatant and pat dry on towel. Resuspend bacterial pellet in 100µl of Buffer P1 (10mM Tris pH8.0, 1mM ETDA pH 8.0, 100µg/ml RNAse A and RNAse T1. Leave for 5' at RT.
  Add 100 µl of Buffer P2 (0.2%SDS, 0.2M NaOH) Leave on ice for 5min
  Add 100µl of 3M Na Acetate pH 4.8 and leave at -20oC for 30min.
  Spin plate at max speed for 60min, 4oC and transfer 125µl to a new 96 well plate. Add 125µl of iso-propanol and precipitate at -20oC for at least one hour. Precipitate plasmid DNAs by spinning the plate at 3000g for 60min at 4oC. Tip out supernatant. Wash with 500µl of 70% Ethanol. Spin 3100rpm for 10min at 4oC.
  Leave plate to dry on bench Resuspend in 50 µl of TE and run 5µl on a gel to check the prep. Dilute 10µl down to 10ng/µl for generating PCR products.
  PCR AMPLIFICATION:
  PCRs are set up as follows:
  Reagent Per Rxn Per 4x 96 Rxns
  --------------------------------------------------------------------------------
  
  10x PCR Buffer 10µl 4.8µl
  25mM MgCl2 7µl 4.0µl
  100mM NH2-M13F* 0.3µl 140µl
  100mM NH2-M13R* 0.3µl 140µl
  5U/µl Taq (AB biotech) 0.8µl 320µl
  H20 to  48ml
  *The primers have a 5' NH2 modified. The primer sequences are:
  M13 FWD: 5' GTT TTC CCA GTC ACG ACG TTG 3'
  M13 REV: 5' TGA GCG GAT AAC AAT TTC ACA CAG 3'
  The bulk mix is then aliquoted into 96 well plates (thermofast, AB biotech). These are skirtless poly-propylene plates. While they don't fit into the arrayer, the have wells deep enough to allow product precipitation and are cheap. Template (5µl) is then added to each reaction. These DNAs had been previously diluted down to approximately 10ng/µl in water.
  Reactions are carried out in a MJ research tetrad PCR machine under the following conditions: (Temperature of block is calculated, plate option is selected, volume of reaction selected is 100µl in the program setup).
  1 x  94oC for 3min
  35 x  48oC for 20sec
  72oC for 3min
  94oC for 40sec
  5µl of the PCR reaction is run out on a 96 lane gel (AB biotech) to determine success of the PCR. PCRs are scored for presence of a product, number of bands and intensity.
  PRODUCT PURIFICATION:
  Isopropanol precipitation:
  Immediately after running out products, add 10µl of 3M Na Acetate (pH5.2) and 100µl iso-propanol. Reseal the plate and precipitate at -20oC overnight. Element is recovered by spinning at 4,100 rpm is approximately 3,100g for 60min at 4oC. Plates are turned over on absorbent towel and gently tapped dry. Pellets are often just visible. Sigma make a rotor that will spin plates at 5,100rpm (approx 4000g) which would be preferable for precipitating these elements.
  Pellets can be washed in 70% ethanol but we have found you can often leave this step out with out obvious problems.
  Leave to air dry for 60min to overnight. Plates are sealed in a storage box and left at -20oC until you are ready to array.
  Prior to arraying, resuspend pellets with the addition of 30µl of H2O and orbital shaking for 60min. Aliquot 10µl material into array working plate. Dry down and resuspend
  Millipore purification derived from Biotechniques, 29(3), Sept 2000,548-562
  Transfer PCR product (50ml) to the Millipore filter plate.
  Place the filter plate on a vacuum manifold filtration system (Qiagen, Cat # 19504 or Millipore Cat. No # MAVM0960R) and filter at a pressure of 15in (380 mm) Hg for 10 minutes or until the plate is dry.
  Add 30ml of MilliQ water to each well and filter at 15mm (380 mm) Hg for 5-10 minutes or until the plate is dry.
  Repeat step 3.
  Remove plate from the manifold filtration system. Add 60ml of MilliQ water to each well and place on a shaker. Shake vigorously for 10 minutes to resuspend the DNA.
  Transfer the purified product to a new 96 well plate.
  Plates containing the purified PCR products are then stored at 4oC for future arraying.
  POST-ARRAY FIXATION:
  Once again, this protocol was adapted from the Brown web site.
  I have taken their protocol and removed the following steps: We have omitted snap drying of the slides. We have omitted post array rehydration.
  Succinic anhydride 6g Aldrich #23,969-0
  1-Methyl-2-pyrrolidinone 325mL Aldrich #32,863-4
  Sodium borate (1M, pH 8) 15mL <= Use boric acid and adjust pH with NaOH
  ddH2O ~1 L
  2L beaker 1
  95% ethanol 350mL Use high grade ethanol
  Mark boundaries of array on back of slide using diamond scriber. Array will become invisible after post-processing.
  UV crosslink DNA to glass with Stratalinker set for 65 mJ. (Set display to "650", which is 650 x 100 mJ.)
  Prepare BLOCKING SOLUTION: Have 3 x 350 ml glass chambers and a large round glass beaker with dH2O ready in the microwave. At this time, prepare the 15ml sodium borate in a 50 mL conical tube.
  Dissolve 6g succinic anhydride in approx. 325-350 mL 1-methyl-2-pyrrolidinone. Rapid addition of reagent is crucial. Immediately after the last flake of the succinic anhydride dissolves, add the 15 mL sodium borate.
  Immediately after sodium borate solution mixes in, pour solution into empty slide chamber. Plunge slide rack rapidly and evenly in solution. Vigorously shake up and down for a few seconds, making sure slides never leave solution.
  Mix on orbital shaker 20 min (50rpm). Meanwhile, heat water 1L of H2O in a microwave (4min max power).
  Gently plunge slide rack in 95oC water (just stopped boiling) for 2 min.
  Plunge slide rack 5X in ice cold (taken out of the -200C freezer) 95% ethanol.
  Centrifuge slides and rack for 2 min. @ 500 rpm at room temperature. Use gloves and forceps when loading to avoid contact with oil and talc.
  Store in slide box. Slides are normally used the following day. Some people recommend leaving at least one week to mature.