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

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>Sam Chiang
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='''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
  line 1. Incubate at 30C for 6 min
  line 2. Plate read
  line 3. Goto line 1 for 99 more times
  line 4. Incubate at 30C for 6 min
  line 5. Plate read
  line 6. Goto line 4 for 99 more times
  line 7. Incubate at 85C for 3 min
  line 8. Incubate at 4C forever
  Note: Tre treatment will delay the reaction time, so we put 200 cycles in total. (The maximum setting is 99 cycles for the 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==
  [[Image:sam031109-N9_Tre.bmp|700px]]
  '''Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction'''
==Discussion==
*It is suggested to do the reaction on ice with rack in stead of the cool block. The cool block is too cold and may frozen the ALS buffer (1 uL) during the cell lysis
*Samples sent to Illumina for genotyping (after 85C, 3 min inactivation)
  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

Latest revision as of 17:17, 18 March 2009