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='''MDA testing using different concentratino of human gDNA template'''= ---- ==Objective== #To repeat the MDA experiment done by Dr. Zhang's on 1-21-09' for contamination test. #This work is done with Jeff for bench work taining purpose as well. ==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 (2 strip) #0.6 mL tubes - for phi29-enzyme master mix #1.5 mL tubes #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: #Label 6 individual PCR tubes and fill with Nuclease-free H2O as following design #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''': # Spin down the tube of N6 primer (261 nmole) # Add 131 uL H2O and mix -> totol conc. = 200 uM # Prepare a fresh 1.5 mL tube. # Perform a 1/200 dilution -> 1 uM N6-primer. Stay on ice for later use. '''Fresh 5M KOH (MW=56.11)''': # Weigh a KOH pellet and put into a fresh 1.5-mL tube. (pellet = 0.078 g = 1.39 x 10^(-3) mole) # Add nucleas-free H2O: 1.39 x 10^(-3) / 5 = 278 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''': # Prepare a 15 mL serological (scre cap) tube. # 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''' # Prepare a 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 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 ''' [[Image:qPCR-sam020609.jpg|600px|none]][[Image:qPCRsam020609-sample annotation.jpg|100px|none]] #'''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)''' #'''F->H: Blank control.''' ==Discussion== #The weak flourescent signle suggested that the amplification was not successul. #The The difference of samples with different template conc. wasn't distinguishable in different curves. #The non-smooth curve may be due to the incomplete dissociation of dsDNA? ==Suggestion== #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). #Check the primer concentration (100 uM for N6-primer should be used?) #Provide enough time for ALS treatment, and shorten the procedure time on after we nutralize the ALS with NS.
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