Hybridisation of fragmented cRNA Things to note: 1. There are two septa on the backside of the GeneChip. Whilst filling or emptying the hybridisation chamber through one septum, a pipette tip must be inserted into the other as a vent. 2. The more you inset/remove tips into the septa, the looser they will become. Eventually they will start to leak. Method 1. Equilibrate array at room temperature for 30 minutes before use. 2. Heat frozen fragmented cRNA at 99oC for 5 minutes. 3. During incubation, pre-wet the array by filling with 200ul (100ul for test or mini arrays) of 1x hybridisation Buffer. Incubate in rotisserie oven at 45oC, 60rpm for 10 minutes. 4. Cool fragmented cRNA at 45oC for 5 minutes. 5. To bring any particulate matter to the bottom of the tube, spin fragmented cRNA at maximum speed (?13,000g) for 5 minutes. 6. Remove buffer solution from array, ensuring that all solution is removed. 7. Add 200ul (100ul for test or mini array) of fragmented cRNA avoiding any debris at the bottom of the tube. 8. Hybridise in rotisserie oven at 45oC, 60rpm, for 16 hours (overnight). 9. Store remaining volume of fragmented cRNA at –20oC. 10. After hybridisation, proceed to “Preparation of GeneChip station”.
Title: Scan with Glasgow Scanner. Description:
HYBRIDISATION Hybridisation control transcripts are added to target cRNA; these include: BioB, BioC and BioD (from the biotin biosynthesis pathway of E. coli) & Cre (from the recombinase gene of bacteriophage P1). A synthetic control oligo (B2) is also added to the hybridisation solution to provide grid alignment signals used by the analysis software. NB: Equilibrate the arrays at RT before starting to prepare the probes. Procedure: 1. Remove 1 vial of the 20 x Eukaryotic hybridisation controls and the vial of oligo B2. 2. Make sure the are 20 x Eukaryotic hybridisation controls completely thawed before proceeding by heating the vials @ 65 C for 5 mins. 3. Lightly vortex the control oligo vials to ensure complete mixing and then briefly spin down the samples. 4. Using the following table, add the appropriate volume of the 20 x Euk Hyb controls and Oligo B2 to your sample. The following table lists the components for the probe (in red the Dros, and all A/B 2.0). Table 12 Component Volume or amount array Type 49 Volume or amount array Type 100 Volume or amount array Type 400 Fragmented cRNA 15 ug 10 ug 5 ug Control Oligo B2 (3 nM) 5 ul 3.3 ul 1.7 ul 20 x Eukaryotic Hyb controls 15 ul 10 ul 5 ul Herring Sperm DNA (10 mg/ml) 3 ul 2 ul 1 ul Acetylated BSA (50 mg/ml) 3 ul 2 ul 1 ul 2 x Hybridisation Buffer 150 ul 100 ul 50 ul DMSO 30 ul 20 ul 10 ul H2O To 300 ul To 200 ul To 100 ul Heat the hybridisation cocktail to 99 C for 5 mins in the PCR machine, followed by 45 C for 5 mins. (AFFY Programme on Eppendorf PCR machine). While hybridisation cocktail is heating wet the array by filling with appropriate volume of 1 x hybridisation buffer (NB remember to fill in upside down position from lower (higher if upright) septum with a 10 ul pipette tip inserted in upper (lower if upright) hole to allow exit of air). Incubate the array @ 45 C for 10 mins in the rotating oven (60 rpm) and ensure that probe array rack is securely clipped in. After incubation spin the hybridisation cocktails @ FS for 5 mins to remove any insoluble material from the hybridisation mixture. Remove the buffer solution from the probe array and fill with the appropriate volume of the clarified hybridisation cocktail avoiding any insoluble matter in the bottom of the tube. Array format Hybridisation Volume (Probe) Total Fill volume (Hyb buffer) 49 200 250 64 200 250 100 (midi) 130 160 169 (mini) 80 100 400 (micro) 80 100 Place probe in box in 45 C oven and rotate @ 60RPM. Hybridise for 16 hours. For standard arrays run the day after the Test3 it is only necessary to thaw probe and proceed from heating the probe up.
PELLET PAINT PRECIPITATION To perform this process several reagents are necessary, they are all listed in table A1. Procedure 1. Add 1ul of pellet paint to each sample 2. Add 0.1 volumes of NaAcO (10% of the sample’s starting volume) 3. Add 2 volumes (200% of the sample’s starting volume) of Absolute EtOH and vortex briefly 4. Incubate @ RT for 2 min 5. Spin sample @ full speed (14,500 rpm) for 5 min. A light blue pellet should be visible @ the bottom of the tube 6. Remove as much as possible of the supernatant 7. Add 500ul of 70% EtOH 8. Spin as in step 5 9. Remove as much as possible of the supernatant 10. Add 500ul of Absolute EtOH 11. Spin as in step 5 12. Remove as much as possible of the supernatant 13. Air dry the pellet for around 5 mins 14. Resuspend the pellet into appropriate volume of RNase free H2O*. *If cDNA resuspend into 12 ul and if tRNA volume to be decided.