User:Vijay/Protocol/microarray

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Overview

Materials

  • Perfect Western (GenHunter Corp)


Procedure

  1. SDS-PAGE and TRANSFER done as per our lab protocol

Buffers

1M KH2P04

(No autoclave, no filter, just make it and use it.... in 100ml glass bottle)
13.6gms in 100ml DEPC water

1M K2HP04

(No autoclave, no filter, just make it and use it... in 100ml glass bottle)
22.8gms in 100ml DEPC water


Phosphate Buffer (1 M KP04, pH ~8.5)

(no autoclave, no filter...just make it and use it in a 50ml falcon tube)
0.5ml 1M KH2P04
9.5ml 1M K2HP04
Total=10ml

Phosphate WASH Buffer (Cloudy)

(no autoclave, no filter...just make it and use it in a 50ml falcon tube)
0.25ml 1M KP04
7.625ml DEPC treated water
42.125ml 95% Ethanol (2.5ml DEPC water + 47.5ml 100% ethanol)
Total=50ml

Phosphate Elution Buffer (4mM)

(no autoclave, no filter... just make it and use it in a 50ml falcon tube)
200ul 1M KP04
to 50ml using DEPC water

POST HYB WASHING

Preheat low stringent buffer to 50C at least for one hour, in the slide washing dish.
Agitate wash slide at rotomix 2.1

  • Low stringent buffer - 5min (50C)
  • Low stringent buffer - 5min (50C)
  • Low stringent buffer - 5min (50C)
  • Medium stringent buffer - 5min (RT)
  • Medium stringent buffer - 5min (RT)
  • High stringent buffer - 10min (RT)
  • High stringent buffer - 10min (RT)
  • Rinse in milliq water
  • Spin ~1000rpm for 10min (RT)