Sam:LabNotes/Microbione/2009-2-4: Difference between revisions

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>Sam Chiang
(New page: ==Objective== # Prepare the primers (human 18S and Bacteria 16S) for in the future MDA experiments (as confirmation) (The Bac 16S primer is based on given sequecne from Dr. Pillai and ...)
 
>Sam Chiang
 
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=Testing human 18S using realtime PCR=
==Objective==
==Objective==
# Prepare the primers (human 18S and Bacteria 16S) for in the future MDA experiments (as confirmation)
#Prepare the primers (human 18S and Bacteria 16S) for the future use in MDA experiments as positive control confirmation (The Bac 16S primer is based on given sequecne from Dr. Pillai and Dr. Dowd's lab. The blat results showed these sequence match most of E. coli genome). Primers info are referred to [[Sam:LabNotes/Microbione/2009-2-3]]
  (The Bac 16S primer is based on given sequecne from Dr. Pillai and Dr. Dowd's lab. The blat results showed these sequence match most of E. coli genome)
#Practice realtime PCR machine operation
# Practice realtime PCR machine operation
#Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme
# Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme


==Reagents==
==Reagents==
Bottled Nuclease-free water (Ambion) - leave in PCR hood for UV decontamination
#H2O - (Ambion, bottled Nuclease-free water) leave in PCR hood for UV decontamination
2X SYBR (working sol.)- diluted from 10,000X (Stock sol.)-> 50X (Stock sol.), only 50X and 2X (diluted) SYBR can be kept in 4 degree
#Dye - 2X SYBR (working sol.)- diluted from 10,000X (Stock sol.)-> 50X (Stock sol.), only 50X and 2X (diluted) SYBR can be kept in 4 degree
2X EconoTaq 2X master - kit  
#Enzyme - EconoTaq 2X master - kit  
  Template - Jerket
#Template - (10 ng/uL)human genome DNA from Jurkat (Human immortalized T-cell line)
#Primer mix (10 uM forward + 10 uM Reverse)
 
==Exp. Design==
  Template         gDNA      H2O
                --------  ---
Primer: 18S-211  #1  #2  #3    (#1, #2 - replicates)
        18S-306  #4  #5  #6    (#4, #5 - replicates)


f1M Tris-HCL (bottled, which company ?)
pH indicator (testing paper)


==Materials==
==Preparation==
strip PCR-tube with caps (2 strip)
1.5 mL tube (x3)
Cleaned Forceps
Cooled tube rack


==Preparing==
'''Primer diluaiton''':
  Making fresh 5M KOH every week (WHY need to be fresh?) MW of KOH = 56.11
  Oligo tube -> add H2O (10 times nmole) uL -> 100 uM
  Preparing 1N HCL (the new bottle is 36%~38% = 12 M = 12 N)
  10 uL of 100 uM (forward) + 10 uL of 100 uM (reverse) + 80 uL H2O => Primer mix
Preparing 1M Tris buffer (the bottled one is 2X, which company ?)
Preparing clean 50X and 2X SYBR Green I solution
Preparing 1 uM N6 primer (diluted from 100 uM)


==Sample==
'''gDNA Template dilution''':
  Human genomic DNA (stored in -20 degree). Stock solution is 10 ng/uL
  Stock solution 100 ug/mL = 100 ng/uL Oligo tube
30 pg/uL = 10 haplotype. 3 pg/uL = 1 haplotype
5 uL of 100 ng/uL + 45 uL H2O -> 50 uL of 10 ng/uL  




==Procedures:==
==Procedures==


===step1: Preparing ALS buffer and NS buffer===
Step1: Preparing master mix
1. ALS buffer:
                              1        (6+1)    rxn
Mix 8 uL 5M KOH, 2 uL 0.5 M EDTA, and 10 uL 1 M DTT with 80 uL nuclease-free H2O, quick spin down.
        --------------------------------------------
        H2O                  7.0      49.0      uL
        SYBR(2X)            1.0        7.0      uL
        EconoTaq 2X Master  10.0       70.0      uL
        --------------------------------------------
                            18.0      126.0      uL(126/7=18)


2. NS buffer:  
Step2: Transfer 18 uL of Master mix into each of 6 individual PCR tubes
Mix 4 mL 1N HCL with 6 mL 1M Tris.HCL (pH = 7.5)


*Make sure that ALS buffer and NS buffer were made correctly, we test the mixture of them (1:1) with pH meter, the pH of mixture should closed to 7~8.
Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design


===step2: Preparing the 2 strips of PCR tubes (for two repeats)===
Step4: Perform real-time PCR  
1. Performing serial dilution for template human gDNA into 30 pg/uL, 3 pg/uL, 300 fg/uL, 30 fg/uL and 3 fg/uL.
        Program name: sam020409
        94C 2min -> (94C 45 sec -> 58C 2 min -> 72C 1 min -> Plate read) x 20 clcle -> 72C 5 min -> 15C hold


*We placed the 45 uL of nuclease-free H2O in PCR tubes first, and transfer 5 uL from the first tube to the second tube.(Transferring: using 20p tips and pippetors; Mixing: using p200 tips and pippetors)
===step3: UV treatment for decontamination of Nucleotide for NS and ALS buffer===
1. In the hood, covering all of gDNA template with black cap first


2. UV treatment for NS and ALS for 10 min
==Results==


===step4: Preparing mater mix for 16+2 reactions===
'''FIG.1 Realtime amplification plot (no log in flourescent)'''
                                1 rxn              18 rxn
[[Image:sam020409.jpg|600px|none]]
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)


===step5: Preparing the reactions===


1. Mix 1 uL template with 1.5 ALS, RT incubation for 3 min
'''FIG.2 Realtime amplification plot (with log in flourescent)'''
[[Image:sam020409-log.jpg|600px|none]]


2. Transfer to cool block (eppendorf)


3. Adding 16 uL Master mix.
==Discussion==


4. Adding 1.5 uL NS
#The amplification curves are not distinguishable (Sample vs. Blank) within 20 clycles.
##The number of amplifications cycles we used is not enough?
##The amplifications are not working? (Primers' specificity? Concentration of templates?)


5. Cap the tube and quick vortexing and spinning down.


6. Place into the realtime machine and start the program.
==Suggestion==
#Perform the Gel electrophoresis for the amplicons -> check the primer specificity
#Repeat the same reactions using regular PCR and bigger cycles(without SYBR)

Latest revision as of 07:20, 9 February 2009

Testing human 18S using realtime PCR[edit]

Objective[edit]

  1. Prepare the primers (human 18S and Bacteria 16S) for the future use in MDA experiments as positive control confirmation (The Bac 16S primer is based on given sequecne from Dr. Pillai and Dr. Dowd's lab. The blat results showed these sequence match most of E. coli genome). Primers info are referred to Sam:LabNotes/Microbione/2009-2-3
  2. Practice realtime PCR machine operation
  3. Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme

Reagents[edit]

  1. H2O - (Ambion, bottled Nuclease-free water) leave in PCR hood for UV decontamination
  2. Dye - 2X SYBR (working sol.)- diluted from 10,000X (Stock sol.)-> 50X (Stock sol.), only 50X and 2X (diluted) SYBR can be kept in 4 degree
  3. Enzyme - EconoTaq 2X master - kit
  4. Template - (10 ng/uL)human genome DNA from Jurkat (Human immortalized T-cell line)
  5. Primer mix (10 uM forward + 10 uM Reverse)


Exp. Design[edit]

Template         gDNA      H2O
                --------  ---
Primer: 18S-211  #1   #2   #3    (#1, #2 - replicates)
        18S-306  #4   #5   #6    (#4, #5 - replicates)


Preparation[edit]

Primer diluaiton:

Oligo tube -> add H2O (10 times nmole) uL -> 100 uM
10 uL of 100 uM (forward) + 10 uL of 100 uM (reverse) + 80 uL H2O => Primer mix 

gDNA Template dilution:

Stock solution 100 ug/mL = 100 ng/uL Oligo tube 
5 uL of 100 ng/uL + 45 uL H2O -> 50 uL of 10 ng/uL 


Procedures[edit]

Step1: Preparing master mix

                             1        (6+1)     rxn
        --------------------------------------------
        H2O                  7.0       49.0      uL
        SYBR(2X)             1.0        7.0      uL
        EconoTaq 2X Master  10.0       70.0      uL
        --------------------------------------------
                            18.0      126.0      uL(126/7=18)

Step2: Transfer 18 uL of Master mix into each of 6 individual PCR tubes

Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design

Step4: Perform real-time PCR

        Program name: sam020409
        94C 2min -> (94C 45 sec -> 58C 2 min -> 72C 1 min -> Plate read) x 20 clcle -> 72C 5 min -> 15C hold


Results[edit]

FIG.1 Realtime amplification plot (no log in flourescent)


FIG.2 Realtime amplification plot (with log in flourescent)


Discussion[edit]

  1. The amplification curves are not distinguishable (Sample vs. Blank) within 20 clycles.
    1. The number of amplifications cycles we used is not enough?
    2. The amplifications are not working? (Primers' specificity? Concentration of templates?)


Suggestion[edit]

  1. Perform the Gel electrophoresis for the amplicons -> check the primer specificity
  2. Repeat the same reactions using regular PCR and bigger cycles(without SYBR)