Sam:LabNotes/Microbione/2009-2-9

From ZhangLabWiki
Revision as of 19:28, 9 February 2009 by >Sam Chiang (New page: =''MDA testing with diluted human gDNA template - revised protocol'''= ---- ==Objective== #To repeat the MDA using correct primer conc. The wrong conc. of N6 primer (1 uM) was used in t...)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

MDA testing with diluted human gDNA template - revised protocol'



Objective

  1. To repeat the MDA using correct primer conc. The wrong conc. of N6 primer (1 uM) was used in the previous version protocol. The correct volume and conc. used in each 10 uL reaction is 1 uL (1 nM), which make the final conc. of N6 primer become 100 uM. using To repeat the MDA experiment done by Dr. Zhang's on 1-21-09' for contamination test.
  2. Prepare the new 2X SYBR

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 (1 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 = 8 rxns)

                                                Sample          Blank    
                                       -----------------------  ---
Template                                30 pg   300 fg   3 fg   H2O    
                                       -----------------------  ---
Primer: repl (using new 2X SYBR)        #A      #B      #C      #D       
        rep2 (using old 2X SYBR)        #A      #B      #C      #D  


Preparation

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

  1. Use the diluted gDNA from 02-06-09' experiments.


2X SYBR Green I dilution:

  1. Dlute the SYBR solution from 10,000X to 2X. Wrap the 2X SYBR with foil and store it in 4 degree.


N6 primer dilution:

  1. Use the diluted 200 uM N6 primer from 02-06-09' experiment.


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. Use the diluted gDNA from 02-06-09' experiments.


phi29 reaction master mix: - for 4+1 rxns

  1. Prepare two 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              5 rxn
         --------------------------------------------------------------
           200 uM N6 primer                10.0 uL              50.0 uL
            10X Repliphi phi-29 buffer      2.0 uL              10.0 uL
            H2O                             1.2 uL               6.0 uL
            25 nM dNTP                      0.8 uL               4.0 uL
            2X SYBR Green I                 1.0 uL               5.0 uL
            Repliphi Phi-29 (100 U/uL)      1.0 uL               5.0 uL
         --------------------------------------------------------------
                                           16.0 uL              80.0 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 order

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

Discussion

Suggestion