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

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(New page: ='''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: ...)
 
>Alan6017518
 
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='''PCR validation for 200pg gDNA limited MDA amplicons-061709 (P1-P12)'''=
='''Genomic DNA Extraction from GM18506 cells'''=
==Samples and Materials==
*3 x 10^6 cells
*Wizard Genomic DNA purification kit
*1.5mL microcentrifuge tubes
*15 mL centrifuge tubes
*PBS
*37C water bath
*Isopropanol
*70% ethanol
*65C water bath


==Procedures==
==Procedure==
*Experiment design
*Harvest the cells and transfer them to a 1.5ml tube
**Primers: Human genome specific primers set 1 (primer 1 to 12)
*Centrifuge at 16000g for 10sec to pellet the cells
**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)
*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)
*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 the 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
 
 
*nanodrop


*Prepare diluted primers in PCR tubes (24 tubes)
='''PCR validation for gDNA'''=
  For each primer needs 7+1 reaction
==sample and material==
 
*human genome specific primers
                            1rxn    (7+1) rxns
*templates
  Primer mix (f+r 10 uM)    1.0      8.0    uL
*Jurkat gdna
  H2O                      7.0    56.0    uL
*h2o
  ---------------------------------------------
*ntc h2o
                            8.0    64.0    uL
*econotaq


==Procedures==
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''GM18506'''
| align="center" style="background:#f0f0f0;"|'''Pos.'''
| align="center" style="background:#f0f0f0;"|'''Neg.'''
|-
| primer mix (f+r 10uM)||1||1||1
|-
| h2o||7||7||9
|-
| econotaq 2x||10||10||10
|-
| Gm template 1ng/ul||2||0||0
|-
| Jurkat template||0||2||0
|-
|
|}


*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.
*Mix well the master mix by vortexing. Transfer 12 uL Master mix into each of 12 reactions.
Line 27: Line 72:
*Transfer 8 uL diluted primer into each reaction based on the arrangements
*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.
*Gently vortex and spin down the PCR reaction tubes. Place in pcr machine immediately to PCR reaction.


*Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 50 -> 72C 10min -> 15C hold
*Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 30 -> 72C 10min -> 15C hold


='''PCR test on the whole set of human genome primers'''=
='''PCR test on the whole set of human genome primers'''=
Line 56: Line 101:
*Loading: Sample 7uL + 6X loading buffer 1.5 uL (Order: P1 -> P24 left to right)
*Loading: Sample 7uL + 6X loading buffer 1.5 uL (Order: P1 -> P24 left to right)
*Run at 135V for 20 min
*Run at 135V for 20 min
[[Image:ZhangLab_2 2009-07-16 14hr 23min pos-test - annotate.jpg|500px]]
*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

Latest revision as of 00:30, 21 July 2009

Genomic DNA Extraction from GM18506 cells[edit]

Samples and Materials[edit]

  • 3 x 10^6 cells
  • Wizard Genomic DNA purification kit
  • 1.5mL microcentrifuge tubes
  • 15 mL centrifuge tubes
  • PBS
  • 37C water bath
  • Isopropanol
  • 70% ethanol
  • 65C water bath

Procedure[edit]

  • 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)
  • 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 the 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


  • nanodrop

PCR validation for gDNA[edit]

sample and material[edit]

  • human genome specific primers
  • templates
  • Jurkat gdna
  • h2o
  • ntc h2o
  • econotaq

Procedures[edit]

' GM18506 Pos. Neg.
primer mix (f+r 10uM) 1 1 1
h2o 7 7 9
econotaq 2x 10 10 10
Gm template 1ng/ul 2 0 0
Jurkat template 0 2 0


  • 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 pcr machine immediately to PCR reaction.
  • Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 30 -> 72C 10min -> 15C hold

PCR test on the whole set of human genome primers[edit]

Background[edit]

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


Procedures[edit]

  • 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[edit]

  • 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