Alice:LabNotes/2010-6-25

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PAGE size selection on Daley's sequencing libraries[edit]

PAGE quantification[edit]

Extract DNA using DNeasy Blood and Tissue kit[edit]

material[edit]

  • 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[edit]

  • 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[edit]

first elution:
CF Fib: 13.4ng/ul * 200ul
second elution:
CF Fib: 4.9ng/ul * 100ul
  • since the concentration is very low, I decided to perform one round of Qiaquick purification on all the DNA that is extracted from CF Fib cells. (including the two tubes of DNA extracted from CF Fib yesterday)
  • total of 600ul of CF Fib DNA (5.2ug total) will be used for Qiaquick column purification
  • Nanodrop result: 67ng/ul * 60ul