NCI60Adherent; RNA harvesting protocol for adherent cells Media used: RPMI 1640 500 ml FBS 25 ml -use the DTP serum if possible, from Bio Whittaker, not heat inactivated 200 mM Glutamine 5 ml 1 flask = ~15 x 106 cells yields ~ 100 ugr RNA. Grow 10 flasks. Cells: start from growing cells from Frederick (not frozen). Started before at passage #8-12. Do not use past passage 20. Growth schedule prior to harvest: Grow cells to ~80 confluencey. Trypsinize cells with 5 ml try-EDTA per T162, 15 min., 370C. Pipet up and down several times w 10 ml pipet to get good dispersment of cells. Count cells. Pass cells to as many flasks as there are cells for. When passing cells, combine flasks into a single pool. Pass 1x106 cells into each T162 w 30 ml media. Repeat growth cycle until 10 flasks are available. Refeed Refeed cells the day prior to harvest without harvesting). Draw off media (wo cells). Add back media, 30 ml per T162. Add back to T-162âs. 37 deg C, ON Harvest Target confluency 80% # of flasks = 10 Draw off media from 1st flask. Lyse cells in 15 ml lysis buffer (w 10 ul fresh BME per ml). Scrape cells. Draw off media from 2nd flask. Pipet lysis buffer from flask 1 into flask 2. Repeat lysis with up to 4 T162âs. Pipet into 50 ml tube. Repeat w next set of 4 flasks. When done, vortex 10 sec.. Draw lysate thru a 20 guage needle 12xâs. Freeze at ö800C. Purify using Quiagen Midi Kit. Use a maximun of 100 x 106 cells per column. More will not bind to the column. Company Info ------------ Quiagen Midi Kit Cat # 75144 RLT Buffer (lysis buffer) Cat. # 79216 $60-list, $51- discounted 1-800-362-7737 Fetal Bovine Serum 500 ml, list $261- Cat #14-502F Lot # 9S083F Cambrex (old Bio Whittaker) 1-800-638-8174 1x PBS pH 7.3-7.5 Bio Whittaker cat #17-516F $5.30 per 500 ml bottle (1-11) 250 ml conical tubes Corning cat #25350-250 RPMI-1640 wo L-glutamine cat # 12-167F $13.25 per 500 ml bottle (for 1-11 bottles) Cambrex (old Bio Whittaker) Walkersville, Md. 21793 301-898-7025 1-800-638-8174 Trypsin (0.05%)-EDTA (0.1%) In phosphate buffered saline without calcium and magnesium. cat # 118-087-061 $4.20 per 100 ml bottle Quality Biological, Inc. 301-840-9331 L-glutamine 200 mM, 100x Gibco-BRL cat # 25030-149 20 ml 1-800-828-6686 162 cm2 flask cat # 3150 Costar Tissue Culture Cell Scrapper, 25 cm Sarstedt Cat. # 83.1830 Cells are received from Nick Scudiero, E-mail: SCUDIERO@dtpax2.ncifcrf.gov Also trypsin, FBS and glutamine have been coming from there as well.; Protocol Type = grow; Parameter start Time = 0; Parameter min temperature = 37; Parameter media = RPMI 1640,5% FBS, 1% L-Glutamine;
Extracted molecule
total RNA
Extraction protocol
NCI60 Extraction Protocol; Harvest Target confluency 80% # of flasks = 10 Draw off media from 1st flask. Lyse cells in 15 ml lysis buffer (w 10 ul fresh BME per ml). Scrape cells. Draw off media from 2nd flask. Pipet lysis buffer from flask 1 into flask 2. Repeat lysis with up to 4 T162’s. Pipet into 50 ml tube. Repeat w next set of 4 flasks. When done, vortex 10 sec.. Draw lysate thru a 20 guage needle 12x’s. Freeze at –800C. Purify using Quiagen Midi Kit. Use a maximun of 100 x 106 cells per column. More will not bind to the column. ; Protocol Type = nucleic_acid_extraction; Parameter Extracted product = total RNA; Parameter Amplification = none;
