Sam:LabNotes/Human/2009-4-12: Difference between revisions

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>Sam Chiang
(New page: ='''Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons'''= ==Procedure== *Experiment design Primer 1-1->6-1 Primer 1-1->6-1 Column ...)
 
>Sam Chiang
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==Procedure==
==Procedure==
*Experiment design
*Experiment design
            Primer 1-1->6-1       Primer 1-1->6-1
**Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1
  Column        B6(3-31)              B7(3-31)     
**Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01


*Thaw following regents:
*Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution)
**Taq pol.(2X)
**Primer Tamplate


*Primers (4 uL)
*Set up realtime PCR program (decribed as following)
  10 uL (10 mM of F + R) + 70 uL H2O mixture
                      1 rxn      20 rxn


    Primer (f+r)        1.0        10 uL
*Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12.
    H2O                7.0        70 uL
    ------------------------------------
                        8.0        80 uL
 
    Place diluted primer in eight PCR tubes(column 1 to 8 (left->right))
    1-1, 2-1, 3-1, 4-1, 1-1, 2-1, 3-1, 4-1


*Aliquots Taq2X enzymes into PCR tubes (100 uL) x 8 PCR tubes
*Expemriment arrangements
 
            Column 1  ->  6        Column 7  -> 12
            Primer 1-1->6-1        Primer 1-1->6-1
                B6(3-31)             B7(3-31)     
                D2(3-31)              D4(3-31) 
                D5(3-31)              D7(3-31) 
                D8(3-31)              B3(4-01) 
                B4(4-01)              B6(4-01) 
                C2(4-01)              C5(4-01) 
                C7(4-01)              C8(4-01) 
                POS(100pg)            NTC 


*Preparing PCR strip tubes
*Prepare diluted primers in PCR tubes
 
                            1rxn  (8+1)x2 ->20rxns
  Primer mix (f+r 10 uM)    1    20 uL
  H2O                        7    140 uL
  --------------------------------------


*Using multi-channel pipette to upload all reagents
*Transfer 8 uL diluted primer into each reaction based on the arrangements
**Enzyme (5 uL): Taq Mix 2X
**Primer (4 uL)


*Using p2.5 pipettor to add diluted (1/50) Templates: (1 uL)
*Transfer 2 uL diluted templates into each reaction based on arrangements
 
  RowA (left) 3-31 B3,  RowA(right) 3-31 B4
  RowB (left) 3-31 B6,  RowB(right) 3-31 C2
  RowC (left) 3-31 C5,  RowC(right) 3-31 C7
  RowD (left) 3-31 C8,  RowD(right) 3-31 gDNA 3 ng


*Program: 52T35C (Tm=52C, for 35 cycles
*Prepare master mix in 1.5-mL microcentrifuge tube
*Gel electrophoresis
                1rxn  (12+2)x8=112 rxns
**2% agarose gel, midium tray with 26-well comb
  Taq (2X)      9.9        1108.8
**Sample (8uL) + Loading buffer (1.5 uL)
  SYBR (50X)    0.1          11.2  
**Run at 135V for 20 min
  --------------------------------
                10.0        1120.0
*Mix well by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.


==Results==
*Gently vortex spin down the PCR reaction tubes. Place in Chromo4 machine to perform realtime PCR reaction.
  [[Image:ZhangLab_2 2009-04-01 14hr 12min-annotate.jpg|700px]]
 
  [[Image:ZhangLab_2 2009-04-01 14hr 52min-annotate.jpg|300px]]
*Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever
 
  Primer(amplicon size)
 
  1-1(270 bp), 2-1(540 bp), 3-1(235 bp), 4-1(383 bp)

Revision as of 01:16, 13 April 2009

Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons

Procedure

  • Experiment design
    • Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1
    • Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01
  • Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution)
  • Set up realtime PCR program (decribed as following)
  • Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12.
  • Expemriment arrangements
            Column 1  ->  6        Column 7  -> 12
            Primer 1-1->6-1        Primer 1-1->6-1
                B6(3-31)              B7(3-31)      
                D2(3-31)              D4(3-31)  
                D5(3-31)              D7(3-31)  
                D8(3-31)              B3(4-01)  
                B4(4-01)              B6(4-01)  
                C2(4-01)              C5(4-01)  
                C7(4-01)              C8(4-01)  
                POS(100pg)            NTC  
  • Prepare diluted primers in PCR tubes
                           1rxn  (8+1)x2 ->20rxns
  Primer mix (f+r 10 uM)     1     20 uL
  H2O                        7    140 uL
  --------------------------------------
  • Transfer 8 uL diluted primer into each reaction based on the arrangements
  • Transfer 2 uL diluted templates into each reaction based on arrangements
  • Prepare master mix in 1.5-mL microcentrifuge tube
               1rxn  (12+2)x8=112 rxns
  Taq (2X)      9.9         1108.8
  SYBR (50X)    0.1           11.2 
  --------------------------------
               10.0         1120.0
  • Mix well by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.
  • Gently vortex spin down the PCR reaction tubes. Place in Chromo4 machine to perform realtime PCR reaction.
  • Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever