AlanFung:LabNotes/Human MDA Halotyping/2009-7-20: Difference between revisions

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==Genomic DNA Extraction from GM18506 cells==
='''Genomic DNA Extraction from GM18506 cells'''=
==Samples and Materials==
*1.5mL microcentrifuge tubes
*15 mL centrifuge tubes
*PBS
*37C water bath
*Isopropanol
*70% ethanol
*65C water bath
 
==Procedure==


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

Revision as of 23:37, 19 July 2009

Genomic DNA Extraction from GM18506 cells

Samples and Materials

  • 1.5mL microcentrifuge tubes
  • 15 mL centrifuge tubes
  • PBS
  • 37C water bath
  • Isopropanol
  • 70% ethanol
  • 65C water bath

Procedure

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