Sam:LabNotes/Microbione/2009-2-26: Difference between revisions

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==Perform MDA on human cell lysates - test 2==
='''Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)'''=
*Samples:
**Fresh lymphocytes, GM18507 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL
**UV-treated (10 min) 1X PBS - 10 mL (prepared in 15-mL Corning tube)




===Lymphocytes pre-processing===
*Take cultured lymphocytes suspension from one T25 flask
**Aliquot 100 uL suspension for cell counting using the cell counter (Beckman)
**Withdraw the desired cell amount (2 x 10^5 cells) and wash using UV-treated 1XPBS twice (Spin at 1000rpm for 3min)
**Resuspend in 1 mL UV-treated PBS


**Make dilution to 10 cell/uL using UV treated PBS:
==Objective==
*Using regular PCR (Taq enzyme 2X) to confirm 8 putative amplicons.


    Dilution Factor    Start      1/10        1/40      1/400   
    ----------------------------------------------------------
    BAC mixture        100 uL    100 uL    10 uL      10 uL
    PBS                            900 uL    30 uL      90 uL
    ----------------------------------------------------------
    Concentration      200/uL      20/uL      5/uL    0.5/uL
 
    Mix by repeat pipetting using p200 pipettor in each step
 
    To decrease the error in the last dilution (1/100 -> 1/1000), this step is performed in four replicates, and labeled #1~#4


==Materials==


*Enzyme - Taq 2X master mix (New England Biolabs)
*Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL
*Human genome primers
**18S-306 (cho. 21) - previously tested
**2-1 (cho.2, 540 bp, Tm 52C)
**3-1 (cho.3, 235 bp, Tm 52C)


*Realtime PCR program set up (Number of reactions)
**cell lysate: 0.5-cell x 24
**Positive controls: 6  No-template control: 2
**Total reaction: 32 x 20ul


*Templates (1 uL): A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1/100 gDNA = 1 ng/uL), H2O


===Set up MDA experiment===


*Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
==Exp. Design==
**Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
         
**Mix 8 ul 5M KOH, 2 ul 0.5M EDTA, 10 ul 1M DTT,  80 ul nuclease free H2O;
                                            Templates (A->H)
**Mix 10ul ALS buffer with 10ul NS buffer, check pH with a pH paper, adjust ALS buffer with KOH or H2O if pH is not within 7-8
            ---------------------------------------------------------------------------
                                A3  A6  A7  B4  B7  C3  C4  C8  p-Pos  p-Neg  Pos  H2O
            ---------------------------------------------------------------------------
  Primer  Strip1 P1 (18S-306)   
            stirp2 P2 (2-1)   
            strip3 P3 (3-1)


==Procedures==
*Thaw the Taq2X enzyme and primers.


*Decontamination with UV-treatment
*Set up 52C program.
**Aliquot ALS buffer and NS buffer to two individual PCR tube, with 100 uL of each buffer.
**Treat the buffer with UV for 10 min.


*Preppare 5 trips of 8-well PCR tubes and transfer the 1 uL template.


*Prepare MDA master mix (in a 1.5-mL tube):                        
*Master Mix - x 5 tubes:
                                1 rxn      x 36 rxn
            1 rxn      14 rxn
  -------------------------------------------------
      ----------------------
  H2O                           6.2 uL  223.2 uL
      H2O     3.5       49.0  
  200 uM N6 primer              5.0 uL  180.0 uL
      Primer  0.5        7.0 - primer is differnt in each one of three master mix
  10x RepliPhi phi-29 buffer    2.0 uL    72.0 uL
      Taq 2X 5.0       70.0
  25mM dNTP                      0.8 uL    28.8 uL
      ----------------------
  2X SYBR Green I                1.0 uL    36.0 uL
              9.0     126.0 (uL)  126/14=
  RepliPhi Phi-29 (100U/ul)      1.0 uL    36.0 uL
  -------------------------------------------------
                                          576.0 uL   (576/36=16)
  Leave on ice for later use
 
*Prepare cell lysates:
**Transfer 1 uL diluted cell (0.5 cell/uL) to each of 32 tubes in four 8-well strips. Diluted cell #1 goes to strip #1, and so on for the rest (#2 -> #2 strip...).


*Transfer 9 uL of master mix into each reaction


*Prepare Pos. and Neg. control:  8 reactions
*Perform PCR reaction
**Transfer 1 uL of 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
**Strip 1 goes to block A (program GENE59: Tm59, 30 cycles)
**gDNA 1 ug/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1)
**Strip 2-3 go to block B (program GENE52: Tm52, 30 cycles)
**DNAse-free H2O (x 2)


*Gel Electrophoresis
**1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> four small tray with 8-well comb)
**Run at 135 V for 20 min.


*DNA denaturing and nutralization:
==Results==
**Add 1.5 uL ALS buffer into 1 uL diluted cells and mix by repeat pipetting (~3 times), incubate at RT for 3 min or until all tubes are finished (Time it!)
**Add 1.5 uL NS buffer, mix by repeat pipetting. Place the tubes on cool rack.


'''FIG. Gel electrophoresis picture '''
  [[Image:ZhangLab_2 2009-02-26 test2 primer_18S.bmp|600px]] [[Image:low mass ladder.bmp|150px]]
  '''Test by primer 18S (306 bp)'''


*Add 16 uL MDA mix to each of the sample tube on cool block. Mix completely by repeat pipetting.
  [[Image:ZhangLab_2 2009-02-26 test2 primer_2-1.bmp|600px]]
  '''Test by primer 2-1 (540 bp)'''


  [[Image:ZhangLab_2 2009-02-26 test2 primer_3-1.bmp|400px]]
  '''Test by primer 3-1 (235 bp)'''


*Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
==Result & Discussion==
 
*Samle B7 and p-Pos contains positive bands by 18S primer (Cho.21/Un) and primer 3-1 (Cho.3).
 
*A very weak band was also found (but not showing clearly in pic) on B7 by primer 2-1 (Cho.2). However the p-Pos failed to show it.
**Sample p-Pos should contain successful amplification. The faliure of p-Pos may be caused by the interference of junk (salt?) left in MDA PCR product.
**Clean the junk using PCR product purification kit and test the PCR again on those p-Pos sample.

Latest revision as of 20:30, 27 February 2009

Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)[edit]

Objective[edit]

  • Using regular PCR (Taq enzyme 2X) to confirm 8 putative amplicons.


Materials[edit]

  • Enzyme - Taq 2X master mix (New England Biolabs)
  • Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL
  • Human genome primers
    • 18S-306 (cho. 21) - previously tested
    • 2-1 (cho.2, 540 bp, Tm 52C)
    • 3-1 (cho.3, 235 bp, Tm 52C)


  • Templates (1 uL): A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1/100 gDNA = 1 ng/uL), H2O


Exp. Design[edit]

                                            Templates (A->H)
           --------------------------------------------------------------------------- 
                                A3  A6  A7  B4  B7  C3  C4  C8  p-Pos  p-Neg  Pos  H2O
           ---------------------------------------------------------------------------
  Primer   Strip1 P1 (18S-306)     
           stirp2 P2 (2-1)    
           strip3 P3 (3-1)

Procedures[edit]

  • Thaw the Taq2X enzyme and primers.
  • Set up 52C program.
  • Preppare 5 trips of 8-well PCR tubes and transfer the 1 uL template.
  • Master Mix - x 5 tubes:
           1 rxn      14 rxn
     ----------------------
     H2O     3.5       49.0 
     Primer  0.5        7.0  - primer is differnt in each one of three master mix
     Taq 2X  5.0       70.0
     ----------------------
             9.0      126.0 (uL)  126/14=9  
  • Transfer 9 uL of master mix into each reaction
  • Perform PCR reaction
    • Strip 1 goes to block A (program GENE59: Tm59, 30 cycles)
    • Strip 2-3 go to block B (program GENE52: Tm52, 30 cycles)
  • Gel Electrophoresis
    • 1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> four small tray with 8-well comb)
    • Run at 135 V for 20 min.

Results[edit]

FIG. Gel electrophoresis picture

  File:ZhangLab 2 2009-02-26 test2 primer 18S.bmp File:Low mass ladder.bmp
  Test by primer 18S (306 bp)
  File:ZhangLab 2 2009-02-26 test2 primer 2-1.bmp
  Test by primer 2-1 (540 bp)
  File:ZhangLab 2 2009-02-26 test2 primer 3-1.bmp
  Test by primer 3-1 (235 bp)

Result & Discussion[edit]

  • Samle B7 and p-Pos contains positive bands by 18S primer (Cho.21/Un) and primer 3-1 (Cho.3).
  • A very weak band was also found (but not showing clearly in pic) on B7 by primer 2-1 (Cho.2). However the p-Pos failed to show it.
    • Sample p-Pos should contain successful amplification. The faliure of p-Pos may be caused by the interference of junk (salt?) left in MDA PCR product.
    • Clean the junk using PCR product purification kit and test the PCR again on those p-Pos sample.