LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
7. Ethidium bromide (10mg/ml)
Method:
A 1.2% reducing agarose gel was prepared by melting 0.36 gm agarose in 25.5
ml DEPC water and cooled down to 55 °C. To this 3 ml 10x MOPS, 1.5 ml
formaldehyde was added and the gel was allowed to set at RT. 1µl of RNA was mixed
with above mentioned RNA loading buffer and heated at 65 °C for 7 min. sample was
allowed to cool and after adding 2 µl of loading dye and 0.2 µl EtBr samples were
loaded on the gel and run at a constant voltage of 50 V for 1.5 h. RNA bands were
visualized and captured on UV exposure on a Gel documentation system.
3.4.3.3 DNase treatment of RNA:
Any downstream application of RNA requires it to be free of DNA
contamination hence β-actin PCR was performed on isolated RNA to ensure any DNA
content and samples were treated with DNase using DNA-free kit wherever required.
Materials:
1. DNA free kit (Ambion)
2. Total RNA
3. Water bath
Method:
For DNase treatment RNA concentration should be ≤200 ng/µl. Total RNA
with ≈200 ng/µl concentration was taken in thin walled 0.6 ml eppendorf tube.
Depending upon the amount of RNA being treated with the enzyme, per 10 µl reaction,
1 µl 10x DNase Buffer and 1 U γ DNase I enzyme was added and the reaction was
incubated at 37 °C for 30 min. To stop the enzyme activity 1 µl DNase inactivator
slurry was added and the reaction was incubated for 2 min at RT with intermittent
mixing so that inactivator can interact with total enzyme in the reaction mixture. The
contents were centrifuged at 10,000 rpm/ RT/ 1.5 min and RNA was transferred to
fresh tube taking care not to pick up the slurry.
3.4.3.4 Preparation of cDNA from total RNA:
cDNA is synthesized in vitro from a RNA template using reverse
transcriptase. This process is called reverse transcription (RT) or first strand cDNA
synthesis. The purpose of converting mRNA to cDNA is mainly for the analysis of the
template mRNA because DNA is much stable than RNA. Once mRNA is converted to
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