Sam:LabNotes/Microbione/2009-2-6

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Revision as of 01:38, 9 February 2009 by >Sam Chiang (→‎Exp. Design)
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MDA testing using different concentratino of human gDNA template


Objective

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


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


Preparing

Making fresh 5M KOH every week (WHY need to be fresh?) MW of KOH = 56.11 
Preparing 1N HCL (the new bottle is 36%~38% = 12 M = 12 N)
Preparing 1M Tris buffer (the bottled one is 2X, which company ?)
Preparing clean 50X and 2X SYBR Green I solution
Preparing 1 uM N6 primer (diluted from 100 uM)


Exp. Design

                            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
Total: 16 rxns

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
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. Preparing 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)


NS buffer:

  1. Preparing 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






Procedures:

step1: Preparing ALS buffer and NS buffer

1. ALS buffer: Mix 8 uL 5M KOH, 2 uL 0.5 M EDTA, and 10 uL 1 M DTT with 80 uL nuclease-free H2O, quick spin down.

2. NS buffer: Mix 4 mL 1N HCL with 6 mL 1M Tris.HCL (pH = 7.5)

  • Make sure that ALS buffer and NS buffer were made correctly, we test the mixture of them (1:1) with pH meter, the pH of mixture should closed to 7~8.

step2: Preparing the 2 strips of PCR tubes (for two repeats)

1. Performing serial dilution for template human gDNA into 30 pg/uL, 3 pg/uL, 300 fg/uL, 30 fg/uL and 3 fg/uL.

  • We placed the 45 uL of nuclease-free H2O in PCR tubes first, and transfer 5 uL from the first tube to the second tube.(Transferring: using 20p tips and pippetors; Mixing: using p200 tips and pippetors)

step3: UV treatment for decontamination of Nucleotide for NS and ALS buffer

1. In the hood, covering all of gDNA template with black cap first

2. UV treatment for NS and ALS for 10 min

step4: Preparing mater mix for 16+2 reactions

                                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)

step5: Preparing the reactions

1. Mix 1 uL template with 1.5 ALS, RT incubation for 3 min

2. Transfer to cool block (eppendorf)

3. Adding 16 uL Master mix.

4. Adding 1.5 uL NS

5. Cap the tube and quick vortexing and spinning down.

6. Place into the realtime machine and start the program.