Etchevers:Notebook/Genomics of hNCC/2009/07/31
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Preparing hNCC for nucleic acid extractions
Alessandro and Marianna both want total RNA, as do we. To prepare it, we've decided to us the miRVana kit AM1560/AM1561 from Ambion/Applied Biosystems.
Samples of 100 to 107 cells per column in 300 μL lysis buffer. (or 0.5 - 250 mg tissue).
RNA quantification by spectrophotometer:
C (concentration in mol/L) = A(260) (e, extinction coefficient in L/mol-cm) (l, path length in cm)
C (in μg/mL) = A(260)/(e.l) x MW (g/moL) x 1000 - for small RNAs under 200nt C is about 33 x A(260) where larger is about 40. Purity want ratio over A(280) 1.8-2.1 .
Simonetta suggests using the Promega RQ1 RNase-free DNase 1U/μL, cat M6101. Will purchase.
7 x T25 en route for the following cultures: R992b, R1066, R1113 and (only three, not thriving) R1107.
Three for Alessandro: proteins for WB, polysome extraction for RNAs, and total RNA from miRVana kit. Two for Marianna: total RNA from miRVana kit, and DNA from QIAamp extraction for eventual methylation analysis. Two for us: total RNA from miRVana kit (perhaps later purification of miRNAs), and one for freezing down again. One extra culture for Sophie for the transfection trials with EGFP.