2. DNase Treatment of Total RNA
1. Reaction solution:
60 ug Total RNA
3 ul RNase Inhibitor
10 ul 10X PCR Buffer
6 ul MgCl2 (25 mM)
20
ul DNase (Promega, 10U/ul)
X ul DEPC Treated water
100 ul total reaction volume
2. Incubate at 37oC for 30 minutes.
3. Add 100 ul Phenol:Chloroform:Iso-Amyl
Alcohol (25:24:1) pH 4.5-5.2
4. Vortex for 30 seconds
5. Spin for 5 minutes at RT at 14,000 RPM.
6. Transfer upper phase (clear solution;
no white goop) to a new microfuge tube.
7. Add:
10 ul of 3 M NaOAc (pH 4.5-5.2)
200 ul 100% ethanol
8. Mix by inverting several times.
9.
Put in DRY ICE/Ethanol bath for 30 minutes or at -80oC for 1 hour.
10. Spin for 15 minutes at 4oC at 14,000 RPM.
11. Remove supernatant
with a pipette very carefully.
12. Resuspend pelleted RNA using pipette in 500 ul of 70 % Ethanol.
13. Spin for 10 minutes at 4oC at
14,000 RPM.
14. Remove supernatant with a pipette.
15. Aspirate remaining drops.
16. Air-dry pellet for 5-10 minutes at RT.
17. Resuspend
RNA pellet in 40 ul DEPC treated water.
18. Spec at OD260 and OD260/280 using 2 ul in 198 ul of water.
Jeffrey D. Randall, Takumi Yoshida,
Ryoji Kojima, Steven Gullans © Page 3 9/6/2001
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans © Page 3 9/6/2001
Total reaction volume 10 ul 20 ul
Add 10 ul of Reaction Buffer to each PCR tube.
Mix well with pipette and incubate at 42oC in PCR machine for 2 hours.
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