I am trying to precipitate RNA isolated from Silkworm larval midgut using absolute and ethanol 3M NaOAc (for storage). The protocol for this has been previously standardized in the lab. It involves re-suspending the RNA in 10 micro litres of DEPC treated water. Since my RNA pellet is quite thick, re-suspension in the mentioned volume makes quantification in biophotometer (Eppendorf) difficult.
Arvind Singh Chandel
GuTC extraction buffer: 4 M guanidine thiocyanate, 1% N-lauroylsarcosine (Na salt, Sarkosyl), 10 mM EDTA, 50 mM HEPES, pH ∼5.3;
Buffer-saturated phenol, pH 4.5-6.6;
Chloroform-isoamyl alcohol mix (24:1);
100% isopropanol (isopropyl alcohol, 2-propanol);
70% ethanol;
10 M LiCl;
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Laurence Stuart Dawkins-Hall
Resuspend RNA initially in100ul of DEPC or molecular grade water (cf certified as RNAse/DNAse free) Then add 1/10 vol 3M sodium acetate pH 4-5 Finalky add 3x vol of ethanol; that is 300ul Now place at -80C
- RNA Precipitated in ethanol at -80C is much more stable than aqueous RNA at -80C
- In terms of reagents you specify I would :
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