Pat Brown's Lab ,Department of Biochemistry and the Howard Hughes Medical Institute
http://cmgm.stanford.edu/pbrown/protocols/3_post_process.html
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Materials for 30 arrays
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Qty
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Order info
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Humid chamber
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1
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Sigma #H 6644
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Inverted heat block (70-80C)
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1
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Diamond scriber
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1
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VWR #52865-005
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Slide rack
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1
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Shandon Lipshaw #121
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Slide chamber
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2
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Shandon Lipshaw #121
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Succinic anhydride
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6 g
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Aldrich #23,969-0
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1-Methyl-2-pyrrolidinone
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325 mL
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Aldrich #32,863-4
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Sodium borate (1M, pH 8)
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15 mL
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<= Use boric acid and adjust pH with NaOH
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ddH2O
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~1 L
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2L beaker
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1
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95% ethanol
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350 mL
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- Mark boundaries of array on back of slide using diamond scriber. Array will become invisible after post-processing.
- Fill bottom of humid chamber with 100 ml 1X SSC.
- Prop slide between two tip boxes with lamp overhead and etch corners of array.
- Place arrays face down over 1X SSC and cover chamber with lid.
- Rehydrate until array spots glisten, approximately 5-15 minutes. Allow spots to swell slightly but not run into each other. Position a lamp ~ 12" overhead.
- Snap-dry each array (DNA side up) on a 70-80C inverted heat block for 3 seconds.
- UV crosslink DNA to glass with Stratalinker set for 65 mJ. (Set display to "650", which is 650 x 100 uJ.)
- Place arrays in slide rack. Have empty slide chamber ready on orbital shaker.
- Prepare BLOCKING SOLUTION:
- Have 3 350 ml glass chambers available (with metal tops) and a large round pyrex dish with dH2O ready in the microwave. At this time, prepare the 15ml sodium borate in a 50 ml conical tube.
- Dissolve 6 g 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 15-20 min. Meanwhile, heat water in pyrex dish (enough to cover slide rack) to boiling.
- Gently plunge slide rack in 95C water (just stopped boiling) for 2 min.
- Plunge slide rack 5X in 95% ethanol.
- Centrifuge slides and rack for 5 min. @ 500 rpm. Load slides quickly and evenly onto the carriers to avoid streaking.
- Use arrays immediately or store in slide box.
Be sure the rack is bent slightly inwards in the middle, or else the slides may run into each other while shaking. 上一篇：Microarray techniques[Stanford UniversityPat ] 下一篇：Design of Yeast Genomic Expression Experiments
