Alice:LabNotes/2010-6-25
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PAGE size selection on Daley's sequencing libraries
PAGE quantification
Extract DNA using DNeasy Blood and Tissue kit
material
- two tubes of cell pallete received from Philip Manos today, and both of them are CF Fib
- going to process one tube of cell pallete
protocol
- resuspend the cell pellete with 800ul of PBS, and take out 200ul (~2.5x10^6 cells) for DNA extraction
a. Resuspend the pellet in 200ul PBS, add 20ul proteinase K 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 yield, repeat last elution step with a new 1.5ml tube
result
first elution:
second elution: