Sam:LabNotes/Microbione/2009-2-6
MDA testing using different concentratino of human gDNA template[edit]
Objective[edit]
- 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[edit]
- 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[edit]
- 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)[edit]
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[edit]
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[edit]
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[edit]
FIG. Quantification of Realtime amplication
- 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[edit]
- 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[edit]
- 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.