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=Extract DNA using DNeasy Blood and Tissue kit= ==material== iPS 29e P21 received from Eggan Lab on 2/12/2010 (cell pellet with unknown cell count) PGP1F received from Jay Lee on 1/27/2010 (cell pellet with 100,000 cells/tube, and one tube is used) ==protocol== a. centrifuge the appropriate number of cells (max 5 million) for 5 min at 1000g. Resuspend the pellet in 200ul PBS, add 20ul proteinase K *put 400ul PBS into iPS cell pellet, break up the cells and aliquot into 2 tubes with 200ul each [RNA-free gDNA: add 4ul RNase A (100mg/ml), mix by vortexing, incubate for 2 min at RT ] b. add 200ul buffer AL (without added ethanol), mix thoroughly by vortexing, and incubate at 56C for 10 min. c. add 200ul 100% ethanol to the sample, and mix thoroughly by vortexing d. pipet the mixture from last step into the DNeasy Mini spin column placed in a 2 ml collection tube. centrifuge at 8000rpm for 1 min, discard the flow-through and collection tube. e. place the DNeasy Mini spin column placed in a new 2 ml collection tube, add 500ul buffer AW1, and centrifuge at 8000rpm for 1 min, discard the flow-through and collection tube. f. place the DNeasy Mini spin column placed in a new 2 ml collection tube, add 500ul buffer AW2, and centrifuge at 14,000rpm for 3 min to dry DNeasy membrane, discard the flow-through and collection tube. h. place the DNeasy Mini spin column in a clean 1.5ml microcentrifuge tube, and pipet 200ul buffer AE on the membrane. Incubate at RT for 1 min, then centrifuge at 8000rpm for 1min i. maximum yeild, repeat last elution step with a new 1.5ml tube ==result== first elution: PGP1F: 95.7ng/ul * 200ul ips 29e P21: 136.6ng/ul * 200ul ips 29e P21: 134.6ng/ul * 200ul second elution: PGP1F: 5.3ng/ul * 100ul ips 29e P21: 64.2ng/ul * 100ul ips 29e P21: 45ng/ul * 100ul
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