Sam:LabNotes/Microbione/2009-2-6

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Revision as of 06:13, 9 February 2009 by >Sam Chiang (→‎Discussion)
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MDA testing using different concentratino of human gDNA template



Objective

  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

  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

  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)

                            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

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

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

FIG. Quantification of Realtime amplication

  1. A->E: Template from high to low conc.
  2. F->H: Blank control.


Discussion

  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

  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.