Introduction to microarray technology Extra material Lecture 17 Statistics 246 March 18 2004 1 Back to cDNA arrays the M Guide Build your own arrayer M Guide Array Maker Documentation Printing Microarrays 2 Printing Microarrays Print Head Plate Handling XYZ positioning Repeatability Accuracy Resolution Environmental Control Humidity Dust Instrument Control Sample Tracking Software 3 Ngai Lab arrayer UC Berkeley 4 Microarray Gridder 5 Slide Preparation Home Grown Protocol for preparing poly L Lysine slides for Microarrays 1 Wash 180 slides completely 2 Prepare poly lysine solution 3 Pour solution over slide 4 Rinse spin dry and store slides 5 Use slides no less than 2 and no more than 4 6 months later 6 7 Product Amplification and preparation What to Print Protocol for Amplifying Products to Print on Array All PCR reactions in 96 well format 100 ml reaction volume Perform PCR reactions in a Tetrad Machine Reactions are assayed on 96 well agarose gel Need multi channel pipetting system Also desirable to have Multimek 96 well pipetting robot 8 MJ Tetrad PCR machine 9 Protocol for preparation of Plasmid DNA from Bacterial Clones Containing Mammalian DNA 1 Inoculate a deep 96 well plate filled with IB antibiotic marker with a small amount of bacterial culture Incubate with shaking at 37 C 2 Spin down the cultures and follow the manufacturer s protocol for the QIAprep 3 Use 1 5 ul of eluted plasmid DNA as PCR template 10 Protocol for precipitation and 384 Well Arraying of PCR products 1 After running reactions on 1 agarose gel and documenting results add sodium acetate pH 5 5 and 110 ul room temp isopropanol 2 Transfer reactions to U bottom plates tape plates together 3 Spin plates at 4 500 rpm for 2 hours 4 Carefully aspirate solution 5 Add 100ul 70 EtOH Spin plates for another hour at 4 500 6 Aspirate again and let air dry or dry in a 96 well speed vac 7 Allow PCR products to resuspend in 20ul of H2O for at least 18 hours 8 Transfer products to 384 well printing plates 9 Dry plates down in speed vac and resuspend products in 3X SSC 10 Let plates resuspend overnight before printing 11 12 Printing Approaches Non Contact Piezoelectric dispenser Syringe solenoid ink jet dispenser Contact using rigid pin tools similar to filter array Tweezer Split pin Micro spotting pin 13 Micro Micro Spotting Spotting pin pin 14 15 Practical Problems Surface chemistry uneven surface may lead to high background Dipping the pin into large volume pre printing to drain off excess sample Spot variation can be due to mechanical difference between pins Pins could be clogged during the printing process Spot size and density depends on surface and solution properties Pins need good washing between samples to prevent sample carryover 16 Post Processing Arrays Protocol for Post Processing Microarrays Hydration Heat Fixing 1 Pick out about 20 30 slides to be processed 2 Determine the correct orientation of slide and if necessary etch label on lower left corner of array side 3 On back of slide etch two lines above and below center of array to designate array area after processing 4 Pour 100 ml 1X SSC into hydration tray and warm on slide warmer at medium setting 5 Set slide array side down and observe spots until proper hydration is achieved 6 Upon reaching proper hydration immediately snap dry slide 7 Place slides in rack 17 Surface blocking 1 Store succinic anhydride in vacuum dessicator until ready for use 2 Measure 335 ml 1 methly 2 pyrrolidinone into designated clean dry slide dish with stir bar 3 Dissolve 5 5 g succinic anhydride completely 4 IMMEDIATELY after succinic anhydride dissolves mix in 15 ml 1M NaBorate pH 8 0 and submerge slides in solution Shake evenly under level of solution 5 Soak slides in solution on shaker for 15 6 Before 15 incubation is done reduce heat on boiling water so temp is approx 95C and no more bubbles are present Drain excess blocking solution off slides and transfer slide rack to boiling water and incubate for 1 30 7 Transfer rack to dish of 95 EtOH and plunge 5X Spin down on tabletop 8 Arrays may be used immediately or stored for future use 18 Isolating Nucleic Acid RNA Membranes and Tumors Protocol for Total RNA isolation in S Cerevisae Modified FastTrack Protocol for Yeast Poly A RNA Isolation Protocol for Poly A Isolations Revised Protocol for FastTrack mRNA extraction from Human Cells Tumor mRNA isolation Gradient based membrane bound Polysome Protocol Protocol for Immunoprecipitation of Chromatin from Fixed Yeast Beadbeater Method 19 20 Protocol for Total RNA Isolation in S Cerevisae 1 Spin down cells about 250ml at OD600 0 5 Dump supernatant 2 Resuspend in 12 ml of AE Buffer Transfer to Oak Ridge phenol resistant centrifuge tubes 3 Add 800 ul 25 SDS 12 ml acid phenol Mix well 4 Incubate 10 at 65 C vortexing every minute 5 Incubate 5 on ice 6 Spin down 15 minutes at 10 000 rpm in SS34 rotor 7 Dump supernatant into pre spun 50 ml PhaseLock tube Add 15 ml chloroform and shake to mix ctd 21 8 Spin down 10 at 3 000 rpm in table top centrifuge 9 Dump supernatant into new oak Ridge tube 10 Add 1 10 volume 3M NaAcetate and equal volume of room temperature isopropanol 11 Spin down 35 40 at 12 000 rpm in SS34 12 Wash with 70 EtOH resuspending pellet spin again 20 at 12 000 rpm 13 Dump off EtOH Dry pellet in vacuum oven briefly 14 Resuspend in 500ul water 15 Quantitate via spec and run 1ug on 1 agarose gel 16 Store total RNA in 80 C Protocol for Poly A Isolations more complex 18 steps 22 Labelling Nucleic Acid Protocol for Reverse transcription and Amino allyl Coupling of RNA Preparation of Fluorescent cDNA Probe from Human mRNA alternate protocol Modified Eberwine ANTISENSE RNA Amplification Protocol Protocol for labeling Genomic DNA for Microarrays Version 1 Genomic DNA Labeling Protocol Round A B DNA Ampification Protocol 23 Preparation of Fluorescent cDNA Probe from Human mRNA alternate protocol 1 To anneal primer mix 2 ug of mRNA with 2 ug of a regular or anchored 5 TTT TTT TTT TTT TTT TTT TT VN 3 oligo dT primer in a total volume of 15 ul x 2 2 Heat to 70 C for 10 min and cool on ice 3 Add 15 ul of reaction mixture each to Cy3 and Cy5 reactions 5X first strand buffer 0 1M DTT unlabeled dNTPs Cy3 or Cy5 Superscript II 4 5X first strand buffer 250 mM Tris HCl 375 KCl 15mM MgCl2 5 Incubate at 42 C for 1 5 2 hrs 6 Degrade RNA by addition of 15ul of 0 1N NaOH and incubate at 70 C ctd 24 7 Neutralize by addition of 15 ul of 0 1N HCl and bring the volume to 500 ul with TE 8 Add 20 ug
View Full Document
Unlocking...