RNA isolation, cDNA synthesis and rtPCR

1 2 3 4 5 6

Analysis of total RNA on denaturing minigel
1. Mix the following ingredients in 125 ml flask.

DEPC-treated

dH2O
10X MOPS agarose

42.5 ml 5ml
0.6 g

2. Heat on stirrer in the fume hood until agarose melts. Cool to 50º C,with stirring.

3. Add 2.55 ml 37% formaldehyde in the fume hood. Pour into gel mold. Insert comb. Allow to cool.

4. Add 20 ul RNA loading buffer to 5 ug totalRNA.

5. Heat to 65-70ºC for 15 minutes to melt secondary structure. Chill on ice. Rapid cooling prevents the formation of
secondary structure.

6. Add 1 ul 1 mg/mg ethidium bromide (EtBr).

7. Flush out gel wells with 1 X MOPS running buffer.
Load samples and run at 40-50 V for 2 hours.

8. Photograph gel using Polaroid camera and UV transilluminator.

@2004 Cebra-Thomas

Last Modified: 17 August 2004

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Analysis of total RNA on denaturing minigel 1. Mix the following ingredients in 125 ml flask.
	DEPC-treated	dH2O 10X MOPS agarose	42.5 ml 5ml 0.6 g
2. Heat on stirrer in the fume hood until agarose melts. Cool to 50º C,with stirring. 3. Add 2.55 ml 37% formaldehyde in the fume hood. Pour into gel mold. Insert comb. Allow to cool. 4. Add 20 ul RNA loading buffer to 5 ug totalRNA. 5. Heat to 65-70ºC for 15 minutes to melt secondary structure. Chill on ice. Rapid cooling prevents the formation of secondary structure. 6. Add 1 ul 1 mg/mg ethidium bromide (EtBr). 7. Flush out gel wells with 1 X MOPS running buffer. Load samples and run at 40-50 V for 2 hours. 8. Photograph gel using Polaroid camera and UV transilluminator.