Sam:LabNotes/Microbione/2009-2-6

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MDA testing using different concentratino of human gDNA template[edit]



Objective[edit]

  1. To repeat the MDA experiment done by Dr. Zhang's on 1-21-09' for contamination test.
  2. This work is done with Jeff for bench work taining purpose as well.


Reagents[edit]

  1. Nuclease free-H2O (Ambion)
  2. 2M Tris-buffer
  3. 1N HCL (diluted from 12N HCL w/w 36%~38%)->1 mL (12N HCL)+ 11 mL H2O -> 12 mL (1N HCL)
  4. 5M KOH (prepare fresh solution in 1.5 mL-tube)
  5. 2X SYBR Green I (diluted from 10,000X stock solution)
  6. 1uM primer
  7. Diluted gDNA


Materials[edit]

  1. strip PCR-tube with caps (2 strip)
  2. 0.6 mL tubes - for phi29-enzyme master mix
  3. 1.5 mL tubes
  4. cool tube rack (Eppendorf) - for reaction preparation


Exp. Design (total = 16 rxns)[edit]

                            Sample                           Blank    
                   -------------------------------------  -------------------
Template           30 pg   3 pg   300 fg   30 fg   3 fg   H2O    H2O    H2O    
                   ----------------------------------------------------------
Primer: repl        #A      #B      #C      #D     #E     #F     #G     #H     
        rep2        #A      #B      #C      #D     #E     #F     #G     #H


Preparation[edit]

Thaw all of the reagents in Phi29 enzyme kit - except the pji29 enzyme"


gDNA Template dilution: Stock solution 100 ug/mL = 100 ng/uL Oligo tube (30 pg/uL = 10 human haplotype. 3 pg/uL = 1 human haplotype) Serial dilutioi:

  1. Label 6 individual PCR tubes and fill with Nuclease-free H2O as following design
  2. Perform the series dilution as followoing design (Note: Transferring by 20p pipettor; Mixing by 200p pipettor)
PCR tubes label                                    1 ng/uL    30 pg/uL     3 pg/uL   300 fg/uL    30 fg/uL     3 fg/uL 
Trasnfer volume             Stock sol. of gDNA ->  1.0 uL  ->   1.5 uL  ->  5.0 uL  ->  5.0 uL  ->  5.0 uL  ->  5.0 uL
H2O in PCR tubes                                  99.0 uL      48.5 uL     45.0 uL     45.0 uL     45.0 uL     45.0 uL 
                                               ----------------------------------------------------------------------
Dilution factor                                    1/100    x   3/100    x  1/10    x   1/10    x   1/10    x   1/10
Conc.                            100 ng/uL         1 ng/uL    30 pg/uL     3 pg/uL   300 fg/uL    30 fg/uL     3 fg/uL


N6 primer dilution:

  1. Spin down the tube of N6 primer (261 nmole)
  2. Add 131 uL H2O and mix -> totol conc. = 200 uM
  3. Prepare a fresh 1.5 mL tube.
  4. Perform a 1/200 dilution -> 1 uM N6-primer. Stay on ice for later use.


Fresh 5M KOH (MW=56.11):

  1. Weigh a KOH pellet and put into a fresh 1.5-mL tube. (pellet = 0.078 g = 1.39 x 10^(-3) mole)
  2. Add nucleas-free H2O: 1.39 x 10^(-3) / 5 = 278 uL
  3. Cap the tube and mix by slight vortexing. Label and date the tube


ALS buffer: - use for dissoicating the dsDNA

  1. Prepare a fresh 1.5-mL tube.
  2. Add 82 uL H2O + 10 uL 1 M DTT + 8 uL 5M KOH in the tube. Mix with quick vortexing and spining down. (Total = 100 uL) (p.s the reagent DTT (1 M) is supplied in the phi29 enzyme kit)


NS buffer:

  1. Prepare a 15 mL serological (scre cap) tube.
  2. Add 3 mL H2O + 3 mL 2M Tris buffer + 4 mL 1N HCL in the tube. Mix with quick vortexing and spining down. (Total = 10 mL)


phi29 reaction master mix: - for 16+2 rxns

  1. Prepare a fresh 0.5 mL tube.
  2. Prepare the master mix as following recipt.
  3. Mix with quick vortexing and spining down (Total = 288 uL ). Leave on ice for later use.


                                            1 rxn              18 rxn
         --------------------------------------------------------------
            H2O                            10.2 uL             183.6 uL
            10X Repliphi phi-29 buffer      2.0 uL              36.0 uL
            1uM N6 primer                   1.0 uL              18.0 uL
            25 nM dNTP                      0.8 uL              14.4 uL
            2X SYBR Green I                 1.0 uL              18.0 uL
            Repliphi Phi-29 (100 U/uL)      1.0 uL              18.0 uL
         --------------------------------------------------------------
                                           16.0 uL             288.0 uL  (288/18=16)


Bio-Rad realtime PCR programing Set up the program as following

         Line 1: 30 C, 6 min
         Line 2: Plate read
         Line 3: Goto line 1 for additional 99 times
         Line 4: 85 C, 3 min (to kill all of the enzymes left)
         Line 5: 4 C, forever
         Line 6: End


Procedures[edit]

Step1: Testing the pH value of NS buffer + ALS buffer mixture (10 uL + 10 uL). The acceptibe pH is between pH 7~8.

Step2: UV both NS and ALS for 10 min for decontamination.

Step3: Prepare 2 stips of PCR tubes and transfer 1 uL template (diluted gDNA or H2O) into each of tubes as exp. design.

Step4: Add 1.5 uL ALS into the tube and mix by pipetting. Incubate at RT for 3 min.

Step5: Add 1.5 uL NS into the tube and mix by pipetting. Transfer the reaction on cool rack.

Step6: Add 16 uL phi29 master mix into each of tubes. Mix by pipetting and quick spining down. Cap and label the tube.

Step7: Place the reaction in Bio-Rad realtime PCR machine and start the reaction program.


Results[edit]

FIG. Quantification of Realtime amplication

  1. A->E: Template from high to low conc. (30 pg/uL, 3 pg/uL, 300 fg/uL, 30 fg/uL and 3 fg/uL, respectively)
  2. F->H: Blank control.

Discussion[edit]

  1. The weak flourescent signle suggested that the amplification was not successul.
  2. The The difference of samples with different template conc. wasn't distinguishable in different curves.
  3. The non-smooth curve may be due to the incomplete dissociation of dsDNA?

Suggestion[edit]

  1. Check the SYBR 2X reagent. The realtime exp on 02-04-09' showed unwanted curves too although the amplification was successful (confirmed by agarose gel).
  2. Check the primer concentration (100 uM for N6-primer should be used?)
  3. Provide enough time for ALS treatment, and shorten the procedure time on after we nutralize the ALS with NS.