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

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>Sam Chiang
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>Sam Chiang
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**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  
**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
   0.093 g (KOH=56.11) = 0.001657 mole;   0.001657/5 x 10^6 =331.49 uL --- H2O needed




Line 43: Line 43:
*Total = 32 rxn -> Prepare (8+1)x4 =36 rxns for master mix
*Total = 32 rxn -> Prepare (8+1)x4 =36 rxns for master mix


 
==Procedures==
*Realtime PCR program set up (Number of reactions)
 
**cell lysate: 0.5-cell x 24
*Thaw the reagents on ice and UV-treated H2O, and equipment for 10 min
**Positive controls: 6  No-template control: 2
**Total reaction: 32 x 20ul


*Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
*Dilute the gDNA in UV-treated H2O into 3ng/uL, 300pg/uL, 30pg/uL, 3pg/uL
**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


*Prepare ALS buffer and test the pH (use old NS buffer is ok)


*Transfer 400 uL(or enough amount) of ALS, NS, 1XPBS, dH2O, into PCR tubes and UV for 10 min


*Decontamination with UV-treatment
*Dilute the cell with UV-treated PBS into 10 cell/uL, 5 cell/uL and 0.5 cell/uL
**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 the 2X SYBR (2uL of 50X SYBR + 48 uL H2O)


*Prepare MDA master mix (in a 1.5-mL tube):                          
*Prepare MDA master mix (in a 0.6-mL tube)and leave on ice                          
                                 1 rxn      x 36 rxn
                                 1 rxn      x 36 rxn
   -------------------------------------------------
   -------------------------------------------------
   H2O                            6.2 uL  223.2 uL
   H2O                            6.2 uL  223.2 uL
   200 uM N6 primer               5.0 uL   180.0 uL
   1mM N9 primer                 1.0 uL   36.0 uL
   10x RepliPhi phi-29 buffer    2.0 uL    72.0 uL
   10x RepliPhi phi-29 buffer    2.0 uL    72.0 uL
   25mM dNTP                      0.8 uL    28.8 uL  
   25mM dNTP                      0.8 uL    28.8 uL  
Line 71: Line 67:
   RepliPhi Phi-29 (100U/ul)      1.0 uL    36.0 uL
   RepliPhi Phi-29 (100U/ul)      1.0 uL    36.0 uL
   -------------------------------------------------
   -------------------------------------------------
                                           576.0 uL  (576/36=16)
                                           432.0 uL  (432/36=12)
   Leave on ice for later use
   Leave on ice for later use
    
    
*Prepare cell lysates:
*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...).
**Trasfer 1 uL ALS buffer into each of 32 PCR tubes on the cool-block
 
**Transfer 1 uL template (Pos, Neg, diluted whole cell) into each of 32 PCR tubes as prevous indication, mix with pipetting in each transferring.
**Incubate for 5 min on cool block
**Transfer 1 uL NS buffer into each of 32 PCR tubes, mix with pipetting.


*Prepare Pos. and Neg. control:  8 reactions
*Transfer 5 uL of Tre(1.2M) into each of 32 PCR tubes, mix with pipetting.
**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)


*Transfer 12 uL MDA master mix to each of 32 PCR tubes. Mix with pipetting.


*DNA denaturing and nutralization:
*Perfomr realtime PCR in Chromo4 with the new program
**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.


*Stop the program (when the reaction reach the signal saturation) and pick up the samples


*Add 16 uL MDA mix to each of the sample tube on cool block. Mix completely by repeat pipetting.
*Inactivate the samples at 85C for 3 min using thermocycler.
 
 
*Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
 




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==Discussion==
==Discussion==


*Nine out of 24 reactions (with 0.5 cell/uL template) were pick up for regular PCR confirmation
*Samples sent to Illumina for genotyping
** A3, A6, A7, B4, B7, C3, C4 and C8
  Sample position      Label              Treatment
 
 
 
                A1      N9-gDNA          (Tre +  N9 primer,  60 pg)
*One Blank reaction (D8) failed due to insufficient Master mix. Need to increase the master mix volume for this design.
                A5      L4N6-gDNA        (Tre +  L4N6 primer, 60 pg)
 
                D2      N9-diluted-lysate (Tre +  N9 primer,   3 pg) 
                E3      N9-whole cell    (Tre +  N9 primer, single cell)
                E5      L4N6-gDNA        (Tre +  L4N6 primer, 3 pg) 
                A9      N9-whole cell    (Tre +  N9 primer, single cell)
                E8      N9-whole cell    (Tre +  N9 primer, single cell)
                G10    N9-whole cell    (Tre +  N9 primer, single cell)


*The back ground amplification (Ct=26~28)still occur in Blank control, althoough the UV-treated H2O (from phi29 kit) was used as template.
                Estimated conc for MDA amplicon:
**ALS and NS buffer were UV-treated. All of the phi29 reagents kit except dNTP and enzyme were UV-treated (10 min).
                For all amplicon by N6 primer - 300 ng/uL
**It is possible the contamination is from the Master mix.
                For all amplicon by N9 primer - 100 ng/uL

Revision as of 20:47, 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

Procedures

  • Thaw the reagents on ice and UV-treated H2O, and equipment for 10 min
  • Dilute the gDNA in UV-treated H2O into 3ng/uL, 300pg/uL, 30pg/uL, 3pg/uL
  • Prepare ALS buffer and test the pH (use old NS buffer is ok)
  • Transfer 400 uL(or enough amount) of ALS, NS, 1XPBS, dH2O, into PCR tubes and UV for 10 min
  • Dilute the cell with UV-treated PBS into 10 cell/uL, 5 cell/uL and 0.5 cell/uL
  • Prepare the 2X SYBR (2uL of 50X SYBR + 48 uL H2O)
  • Prepare MDA master mix (in a 0.6-mL tube)and leave on ice
                                1 rxn      x 36 rxn
  -------------------------------------------------
  H2O                            6.2 uL   223.2 uL
  1mM N9 primer                  1.0 uL    36.0 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
  -------------------------------------------------
                                          432.0 uL   (432/36=12)
  Leave on ice for later use


  • Prepare cell lysates:
    • Trasfer 1 uL ALS buffer into each of 32 PCR tubes on the cool-block
    • Transfer 1 uL template (Pos, Neg, diluted whole cell) into each of 32 PCR tubes as prevous indication, mix with pipetting in each transferring.
    • Incubate for 5 min on cool block
    • Transfer 1 uL NS buffer into each of 32 PCR tubes, mix with pipetting.
  • Transfer 5 uL of Tre(1.2M) into each of 32 PCR tubes, mix with pipetting.
  • Transfer 12 uL MDA master mix to each of 32 PCR tubes. Mix with pipetting.
  • Perfomr realtime PCR in Chromo4 with the new program
  • Stop the program (when the reaction reach the signal saturation) and pick up the samples
  • Inactivate the samples at 85C for 3 min using thermocycler.


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

  • Samples sent to Illumina for genotyping
  Sample position      Label              Treatment 
 
               A1      N9-gDNA           (Tre +  N9 primer,   60 pg)
               A5      L4N6-gDNA         (Tre +  L4N6 primer, 60 pg)
               D2      N9-diluted-lysate (Tre +  N9 primer,    3 pg)   
               E3      N9-whole cell     (Tre +  N9 primer, single cell)
               E5      L4N6-gDNA         (Tre +  L4N6 primer,  3 pg)  
               A9      N9-whole cell     (Tre +  N9 primer, single cell)
               E8      N9-whole cell     (Tre +  N9 primer, single cell)
               G10     N9-whole cell     (Tre +  N9 primer, single cell)
               Estimated conc for MDA amplicon:
                For all amplicon by N6 primer - 300 ng/uL
                For all amplicon by N9 primer - 100 ng/uL