Sam:LabNotes/Microbiome/2009-3-10: Difference between revisions

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>Sam Chiang
(New page: ='Perform MDA on human gDNA, diluted whole cells'''= ==Sample & Material== *Cell: GM18506 cell line, harvested, washed by UV-treated PBS and coundted to 200,000 cell/mL (200 cell/uL) **D...)
 
>Sam Chiang
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='Perform MDA on human gDNA, diluted whole cells'''=
='''Perform MDA on human gDNA, diluted whole cells'''=
 
 
==Objective==
*Based on previous exp, the N9 primer plus Tre (1.2 M) treatmen can provide the best result with highest accuration on correct calls). The current experiment is designed to capture the diluted whole cell using the same set up.


==Sample & Material==
==Sample & Material==
*Primer:N9 primer


*Cell: GM18506 cell line, harvested, washed by UV-treated PBS and coundted to 200,000 cell/mL (200 cell/uL)
*Cell: GM18506 cell line, harvested, washed by UV-treated PBS and coundted to 200,000 cell/mL (200 cell/uL)
Line 12: Line 17:


                              
                              
*Pos. control: human gDNA diluted to 3 ng/uL
*Pos. control: human gDNA diluted to 3 ng/uL
**Measure the original (commercial human gDNA) using Nanodrop -> 309ng/uL
**Measure the original (commercial human gDNA) using Nanodrop -> 309ng/uL
Line 20: Line 24:
   Transfering(uL)          10          10          10        10                     
   Transfering(uL)          10          10          10        10                     
   UVed-PBS(uL)                    90          90        90        90
   UVed-PBS(uL)                    90          90        90        90
*Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
**Prepare 5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH.
**Mix 20 ul 5M KOH, 5 ul 0.5M EDTA, 25 ul 1M DTT(Qiagen), 200 ul nuclease free H2O '''(Note: this is new formula)'''
**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
  0.093 g (KOH=56.11) = 0.001657 mole  0.001657/5 x 10^6 =331.49 uL --- H2O needed
==Reaction arrangement==
*Left(A) to Right(H) 
**Row1: 3ng, 300pg, 30pg, 3pg x 3, H2O x 2
**Row2: 0.5 cell x 8
**Row3: 0.5 cell x 8
**Row4: 0.5 cell x 6, 10 cell, 5 cell
*Total = 32 rxn -> Prepare (8+1)x4 =36 rxns for master mix
 
*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
*Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
**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;
**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
*Decontamination with UV-treatment
**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.
*Prepare MDA master mix (in a 1.5-mL tube):                       
                                1 rxn      x 36 rxn
  -------------------------------------------------
  H2O                            6.2 uL  223.2 uL
  200 uM N6 primer              5.0 uL  180.0 uL
  10x RepliPhi phi-29 buffer    2.0 uL    72.0 uL
  25mM dNTP                      0.8 uL    28.8 uL
  2X SYBR Green I                1.0 uL    36.0 uL
  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...).
*Prepare Pos. and Neg. control:  8 reactions
**Transfer 1 uL of 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
**gDNA 1 ug/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1)
**DNAse-free H2O (x 2)
*DNA denaturing and nutralization:
**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.
*Add 16 uL MDA mix to each of the sample tube on cool block. Mix completely by repeat pipetting.
*Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
==Results==
  [[Image:sam022509-graph only2-with annotation.bmp|600px]]
  '''Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction'''
==Discussion==
*Nine out of 24 reactions (with 0.5 cell/uL template) were pick up for regular PCR confirmation
** A3, A6, A7, B4, B7, C3, C4 and C8
*One Blank reaction (D8) failed due to insufficient Master mix. Need to increase the master mix volume for this design.
*The back ground amplification (Ct=26~28)still occur in Blank control, althoough the UV-treated H2O (from phi29 kit) was used as template.
**ALS and NS buffer were UV-treated. All of the phi29 reagents kit except dNTP and enzyme were UV-treated (10 min).
**It is possible the contamination is from the Master mix.

Revision as of 19:03, 11 March 2009

Perform MDA on human gDNA, diluted whole cells

Objective

  • Based on previous exp, the N9 primer plus Tre (1.2 M) treatmen can provide the best result with highest accuration on correct calls). The current experiment is designed to capture the diluted whole cell using the same set up.

Sample & Material

  • Primer:N9 primer
  • Cell: GM18506 cell line, harvested, washed by UV-treated PBS and coundted to 200,000 cell/mL (200 cell/uL)
    • Dilute to 10 cell/uL, 5 cell/uL, 0.5 cell/uL using UV-treated PBS
  Dilute factor     original    x 1/2   x 1/10   x 1/2    x 1/10
  conc.(cell/uL)      200/uL   100/uL    10/uL    5/uL    0.5/uL
  Transfering(uL)           50        10       50      10                     
  UVed-PBS(uL)                   50       90       50      90


  • Pos. control: human gDNA diluted to 3 ng/uL
    • Measure the original (commercial human gDNA) using Nanodrop -> 309ng/uL
  Dilute factor    original      x 1/10       x 1/10     x 1/10    x 1/10
  conc.            309ng/uL      3ng/uL     300pg/uL    30pg/uL    3pg/uL
  Transfering(uL)           10           10          10         10                     
  UVed-PBS(uL)                     90           90         90         90


  • Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
    • Prepare 5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH.
    • Mix 20 ul 5M KOH, 5 ul 0.5M EDTA, 25 ul 1M DTT(Qiagen), 200 ul nuclease free H2O (Note: this is new formula)
    • 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
  0.093 g (KOH=56.11) = 0.001657 mole   0.001657/5 x 10^6 =331.49 uL --- H2O needed


Reaction arrangement

  • Left(A) to Right(H)
    • Row1: 3ng, 300pg, 30pg, 3pg x 3, H2O x 2
    • Row2: 0.5 cell x 8
    • Row3: 0.5 cell x 8
    • Row4: 0.5 cell x 6, 10 cell, 5 cell
  • Total = 32 rxn -> Prepare (8+1)x4 =36 rxns for master mix


  • 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
  • Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
    • 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;
    • 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


  • Decontamination with UV-treatment
    • 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.


  • Prepare MDA master mix (in a 1.5-mL tube):
                                1 rxn      x 36 rxn
  -------------------------------------------------
  H2O                            6.2 uL   223.2 uL
  200 uM N6 primer               5.0 uL   180.0 uL
  10x RepliPhi phi-29 buffer     2.0 uL    72.0 uL
  25mM dNTP                      0.8 uL    28.8 uL 
  2X SYBR Green I                1.0 uL    36.0 uL
  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...).


  • Prepare Pos. and Neg. control: 8 reactions
    • Transfer 1 uL of 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
    • gDNA 1 ug/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1)
    • DNAse-free H2O (x 2)


  • DNA denaturing and nutralization:
    • 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.


  • Add 16 uL MDA mix to each of the sample tube on cool block. Mix completely by repeat pipetting.


  • Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.


Results

  File:Sam022509-graph only2-with annotation.bmp
  Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction

Discussion

  • Nine out of 24 reactions (with 0.5 cell/uL template) were pick up for regular PCR confirmation
    • A3, A6, A7, B4, B7, C3, C4 and C8


  • One Blank reaction (D8) failed due to insufficient Master mix. Need to increase the master mix volume for this design.


  • The back ground amplification (Ct=26~28)still occur in Blank control, althoough the UV-treated H2O (from phi29 kit) was used as template.
    • ALS and NS buffer were UV-treated. All of the phi29 reagents kit except dNTP and enzyme were UV-treated (10 min).
    • It is possible the contamination is from the Master mix.