Sam:LabNotes/Microbione/2009-2-4
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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 Dr. Dowd's lab. The blat results showed these sequence match most of E. coli genome) # Practice realtime PCR machine operation # Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme
Reagents
Bottled Nuclease-free water (Ambion) - 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 2X EconoTaq 2X master - kit Template - Jerket
f1M Tris-HCL (bottled, which company ?)
pH indicator (testing paper)
Materials
strip PCR-tube with caps (2 strip) 1.5 mL tube (x3) Cleaned Forceps Cooled tube rack
Preparing
Making fresh 5M KOH every week (WHY need to be fresh?) MW of KOH = 56.11 Preparing 1N HCL (the new bottle is 36%~38% = 12 M = 12 N) 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
Human genomic DNA (stored in -20 degree). Stock solution is 10 ng/uL
30 pg/uL = 10 haplotype. 3 pg/uL = 1 haplotype
Procedures:
step1: Preparing ALS buffer and NS buffer
1. ALS buffer: 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.
2. NS buffer: 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.
step2: Preparing the 2 strips of PCR tubes (for two repeats)
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.
- 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
step4: Preparing mater mix for 16+2 reactions
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)
step5: Preparing the reactions
1. Mix 1 uL template with 1.5 ALS, RT incubation for 3 min
2. Transfer to cool block (eppendorf)
3. Adding 16 uL Master mix.
4. Adding 1.5 uL NS
5. Cap the tube and quick vortexing and spinning down.
6. Place into the realtime machine and start the program.