Preparation of Dendrimer Cy3 and Cy5.
Prepare a mixture of Cy3 dendrimer and Cy5 dendrimer in a 1:1 ratio.
(a stock solution
of 36 ul (18ul each) can be made and kept in a light block tube at +4oC)
Incubate the dendrimer mixture at 65oC for 10 minutes.
Vortex
dendrimer mixture for 30-60 seconds to break up aggregates.
Incubate at 65oC until use (Should be about 10 more minutes).
Denature the
probe at 95oC for 3-5 minutes in PCR machine.
Transfer immediately to 65oC waterbath.
Add:
3 ul Dendrimer mix to each probe and mix well.
Incubate
at 65oC for 30 minutes to 1 hour to facilitate probe-dendrimer hybridization.
Add:
Using the
Continue loading all probes into respective chambers.
Hybridize O/N.
200 ul ExpressHyb
to each probe and mix well.
Quick spin down and return to 65oC.
Flush with 2 ml of 2X SSC + 0.2% SDS at 55oC.
Flush with 2 ml of 1X SSC at Room Temp.
Flush with 2 ml of 0.1X
SSC at Room Temp.
Air Dry slides for 120 seconds each. END PROGRAM.
Remove slides from chambers and place in stainless steel slide rack.
Spin
for 1 minute at 500-1000 rpm in Jouan centrifuge.
SCAN in ScanArray 5000.
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans
?Page 5 9/6/2001
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans ?Page 5 9/6/2001
6. Processing of Array slide
Place
the spotted chip on plastic tray (DNA SIDE UP).
Cross-link the DNA to the chip using Stratagene Stratalinker.
Turn Power on.
Press RESET
Press
ENERGY
Press 5500
Press START
Put slide in 80oC oven for two hours (DNA SIDE UP).
Using slide holder, place arrays in 0.2% SDS for 10
minutes with gentle mixing/stirring.
Wash the arrays in R/O water 2x.
Place arrays in plastic container for 2 minutes in fresh R/O water.
Transfer
the arrays to 100% ethanol (?5oC) for 2 minutes.
Spin slides for 1 minute at 500-1000 RPM in Jouan centrifuge.
Dry slide at RT and place
in light block slide box.
Microarray Protocols
Basic knowledge of microarray.
Basic knowledge of microarray 2.
MicroArray Procedure
(Modified by Ryoji Kojima, Takumi Yoshida and Jeffrey Randall,
8.20.2000)
1. Total RNA Isolation from cultured cells.
2. DNase Treatment of Total RNA
3. Making the single strand
cDNA probe.
4. Automated Slide Processor (ASP) Version for hybridization.
5. Washing microarrays in ASP.
7. Pre-hybridization of the processed slides (NON-Automated version).
8. Hybridization of Cy3 + Cy5 probe
to glass array (NON-Automated version).
9. Preparation of Dendrimer Cy3 and Cy5.
10. Washing unbound probe from glass array
(NON-Automated version).
11. Hybridization of Dendrimers (Cy3 and Cy5) to Array (NON-Automated version).
12.Washing unbound
dendrimer from glass array(NON-Automated version).
Other Microarray Protocols (1)
Other Microarray Protocols (2)
Web Guider
Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics