AlanFung:LabNotes/Human MDA Halotyping/2009-7-20

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Revision as of 14:53, 20 July 2009 by >Alan6017518 (→‎Procedure)
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Genomic DNA Extraction from GM18506 cells

Samples and Materials

  • Wizard Genomic DNA purification kit
  • 1.5mL microcentrifuge tubes
  • 15 mL centrifuge tubes
  • PBS
  • 37C water bath
  • Isopropanol
  • 70% ethanol
  • 65C water bath

Procedure

  • Harvest the cells and transfer them to a 1.5ml tube
  • Centrifuge at 16000g for 10sec to pellet the cells
  • Remove the supernatant
  • Add 200ul pbs to wash the cells repeat centrifuge and remove pbs
  • Vortex vigorously to resuspend cells?????
  • Add 600 ul of nuclei lysis solution and pipet to lyse the cells (pipet until no visible cell clumps remain)
  • OPTIONAL add 3ul of rnase solution to the nuclear lysate and mix the sample by inverting the tube 5 times uncubate the mixture for 30m 37C allow the sample to cool to rt for 5m before proceeding
  • add 200ul of protein precipitation solution and vertex vugorously at high speed for 20sec
  • Chill sample on ice for 5m
  • centrifuge for 4n at 16000g
  • add 600ul rt isopropanol to a 1.5ml tube
  • transfer the supernatant containing the DNA to the 1.5ml tube
  • gently mix the solution by inversion until the white thread like strands of dna form a visible mass
  • centrifuge for 1m at 16000g at rt
  • decant the supertant
  • add 600ul of rt 70% ethanol and gently invert the tube several times to wash the dna
  • centrifuge for 1m at 16000g at rt
  • decant the supernatant
  • invert the tube on clean absorbent paper and air dry h pellet for 15m
  • add 100ul of dna rehydration solution
  • incubate at 65C for 1 hr periodialy mix the solution by gently tapping the tube
  • store dna at 4C

PCR validation for 200pg gDNA limited MDA amplicons-061709 (P1-P12)

Procedures

  • Experiment design
    • Primers: Human genome specific primers set 1 (primer 1 to 12)
    • Templates: 200pg MDA amplicons from 06/16/09 200pg MDA: L4N7, N6 in 1/50 dilution. Pos (100 pg/uL gDNA), NTC (RT-PCR grade H2O)
  • Prepare diluted primers in PCR tubes (24 tubes)
  For each primer needs 7+1 reaction
 
                            1rxn    (7+1) rxns
  Primer mix (f+r 10 uM)    1.0      8.0    uL
  H2O                       7.0     56.0    uL
  ---------------------------------------------
                            8.0     64.0    uL


  • Prepare master mix in 1.5-mL microcentrifuge tube, wrapped with foil
                   1rxn        (12+2) rxns x 7
   Econo (2X)      10.0         140.0
   Template         2.0          28.0 – (N6, 15C, 20C, 1-100dNTP, 1-1000dNTP, Pos, H2O)
  -----------------------------------
                   12.0         168.0	 168/14=12
  • Mix well the master mix by vortexing. Transfer 12 uL Master mix into each of 12 reactions.
  • Transfer 8 uL diluted primer into each reaction based on the arrangements
  • Gently vortex and spin down the PCR reaction tubes. Place in Chromo4 machine immediately to perform realtime PCR reaction.
  • Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 50 -> 72C 10min -> 15C hold

PCR test on the whole set of human genome primers

Background

  • Testing if the human genome primers (f+r 10uM premixed) were degraded.


Procedures

  • Primers: Human genome specific primers set 1 (primer 1 to 24)
  • Templates: Pos (1 ng/uL gDNA)
                            1rxn  
  Primer mix (f+r 10 uM)    1.0     uL
  H2O                       7.0     uL
  Template                  2.0     uL
  Econo (2X)               10.0     uL
  ---------------------------------------------
                           20.0     uL

  Amplified at 52C for 40 cycles

Gel electrophoresis

  • 110mL (0.5X TBE) 2% agarose + 8 uL SYBR safe -> 2 middle gel with 26-well comb
  • Loading: Sample 7uL + 6X loading buffer 1.5 uL (Order: P1 -> P24 left to right)
  • Run at 135V for 20 min
File:ZhangLab 2 2009-07-16 14hr 23min pos-test - annotate.jpg


  • All of the human genome primers are still working fine. 40 cycles amplification with 7uL sample loading amount will be used as the standard for following PCR tests