Edited by faxinsd, 27 June 2004 - 05:57 PM.
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2 replies to this topic
#2
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Posted 27 June 2004 - 07:44 PM
Hi.
you should firstly precipitate mRNA with 1/10 th volume 3M Sodium Acetate, 1/50 volume Glycogen(5mg/ml), and 3 volume ice cold 100% ethanol at -20C for at least 1hr, then resuspend the precipitate in TE buffer(pH 8.0) .
Good luck
weimin
you should firstly precipitate mRNA with 1/10 th volume 3M Sodium Acetate, 1/50 volume Glycogen(5mg/ml), and 3 volume ice cold 100% ethanol at -20C for at least 1hr, then resuspend the precipitate in TE buffer(pH 8.0) .
Good luck
weimin
#3
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Posted 27 June 2004 - 07:46 PM
Here is a protocol used to concentrate poly(A)+ mRNA:
1. Add 1/10 volume 3 M NaAc, pH 5.2, and 2.5 and 2.5 volumes ethanol.
2. Mix and incubate at -20C for at least 1 hour.
3. Centrifuge at > 12,000 x g in a microfuge for 20 minutes at 4C.
4. Wash the pellet twice with 80% ethanol.
5. Air dry pellet. Check for dryness before proceeding.
6. Resuspend pellet in RNase-, DNase-free H2O. The appropriate volume for resuspension depends on the expected yield and the amount of RNA required for the cDNA synthesis.
Reference: Affymetrix Expression Analysis Technical Manual.
1. Add 1/10 volume 3 M NaAc, pH 5.2, and 2.5 and 2.5 volumes ethanol.
2. Mix and incubate at -20C for at least 1 hour.
3. Centrifuge at > 12,000 x g in a microfuge for 20 minutes at 4C.
4. Wash the pellet twice with 80% ethanol.
5. Air dry pellet. Check for dryness before proceeding.
6. Resuspend pellet in RNase-, DNase-free H2O. The appropriate volume for resuspension depends on the expected yield and the amount of RNA required for the cDNA synthesis.
Reference: Affymetrix Expression Analysis Technical Manual.
