Sam:LabNotes/Microbione/2009-2-6: Difference between revisions
>Sam Chiang (New page: ='''MDA testing using different concentratino of human gDNA template'''= ---- ==Reagents== Nuclease free water – for serial dilution (Ambion) 2M Tris-buffer ( which company ?) ==Ma...) |
>Sam Chiang |
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='''MDA testing using different concentratino of human gDNA template'''= | ='''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== | ==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== | ==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. | |||
2. | 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. | |||
1. | |||
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. |
Latest revision as of 08:14, 9 February 2009
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.