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='''MDA testing with diluted human gDNA template - revised protocol'''= ---- ==Objective== #To repeat the MDA using correct primer concentration. Wrong concentrtion of N6 primer was used in the previous version protocol. The correct volume and concentration used in each 20 uL reaction should be 1 uL of 1 nM, which make the final conc. of N6 primer become 50 uM. #Prepare the new 2X SYBR reagents and compare with old 2X SYBR. Check if the old 2X SYBR is out of function. ==Reagents== #Nuclease free-H2O (Ambion) #2M Tris-buffer #1N HCL (diluted from 12N HCL w/w 36%~38%)->1 mL (12N HCL)+ 11 mL H2O -> 12 mL (1N HCL) #5M KOH (prepare fresh solution in 1.5 mL-tube) #2X SYBR Green I (diluted from 10,000X stock solution) #1uM primer #Diluted gDNA ==Materials== #strip PCR-tube with caps (1 strip) #0.6 mL tubes - for phi29-enzyme master mix #1.5 mL tubes #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 phi29 enzyme''' '''Human Jurkat cell gDNA dilution''' # Prepare the gDNA dilution as described in 02-06-09' experiment. # Only 30 pg/uL, 300 fg/uL and 3 fg/uL were used in this exp. '''2X SYBR Green I dilution''': # Dlute the SYBR solution from 10,000X to 2X. Wrap the 2X SYBR with foil and store it in 4 degree. '''N6 primer dilution''': # Use the diluted 200 uM N6 primer from 02-06-09' experiment. '''Fresh 5M KOH (MW=56.11)''': # Weigh a KOH pellet and put into a fresh 1.5-mL tube. pellet = 0.065 g = 1.158 x 10^(-3) mole # Add nucleas-free H2O: 1.158 x 10^(-3) / 5 = 232 uL # Cap the tube and mix by slight vortexing. Label and date the tube '''ALS buffer''': - use for dissoicating the dsDNA # Prepare a fresh 1.5-mL tube. # 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''': # Use the one from 02-06-09' experiments. '''phi29 reaction master mix: - for 4+1 rxns''' # Prepare two fresh 0.5 mL tube. # Prepare the master mix as following recipt. # Mix with quick vortexing and spining down (Total = 288 uL ). Leave on ice for later use. 1 rxn 5 rxn -------------------------------------------------------------- 200 uM N6 primer 5.0 uL 25.0 uL 10X Repliphi phi-29 buffer 2.0 uL 10.0 uL H2O 6.2 uL 31.0 uL 25 nM dNTP 0.8 uL 4.0 uL 2X SYBR Green I 1.0 uL 5.0 uL --(New and old 2X SYBR for master mix 1 and 2, respectively) Repliphi Phi-29 (100 U/uL) 1.0 uL 5.0 uL -------------------------------------------------------------- 16.0 uL 80.0 uL (80/5=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. Cap and label the tube. Step7: Place the reaction in Bio-Rad realtime PCR machine and start the reaction program. ==Results== '''FIG. 1 Amplification curve - Using new 2X SYBR (rep1) ''' [[Image:sam020909-rep1.jpg|400px|none]] '''FIG. 2 Amplification curve - Using old 2X SYBR (rep2) ''' [[Image:sam020909-rep2.jpg|400px|none]] ==Discussion== #The curves of rep1 show difference between differnt conc. of diluted templated with successful amplications. #The results of rep2 suggest that old 2X SYBR may have contamination or in wrong conc. that can interfere the amplication. Similarly the interfered amplication using old 2X SYBR was observed in previous realtime PCR test (02-03-09').
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