Sam:LabNotes/Microbiome/2009-3-9 exp2
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Multiple PCR against human whole genomoe[edit]
Objective[edit]
- Based on the previous multiple PCR result, increase the primer concentration for those weak bands
- Test the effect of (DMSO-like) Q-solution come with Qiagen multiple PCR kit
Material and Samples[edit]
Template: human gDNA (1 ng/uL)
- Prepare the new primer gourp mix for primer set-1(1X = 10 uL, 2X = 20 uL)
- Set-1 ("B") Blue: including Y-1(2X), 21-1(2X), the rest primers (1X)
- Set-1 ("Y") Yellow: 11-1(2X), 10-1(2X), 16-1(2X)
- Set-1 ("R") Red: Use the old one
- Set-1 ("G") Green: 18-1(2X), 3-1(2X)
- Set-1 ("O") Orange: 20-1(2X), 7-1(2X)
Procedures[edit]
"M": Master mix (no Q-solution) 1 rxn 6 rxn -------------------------- H2O 15.0 90.0 Template 5.0 30.0 Taq2X 25.0 150.0 -------------------------- 45.0 270.0 (uL) 270/6=45
"MQ": Master mix (with Q-solution) 1 rxn 6 rxn -------------------------- Q-sol 5.0 30.0 H2O 10.0 60.0 Template 5.0 30.0 Taq2X 25.0 150.0 -------------------------- 45.0 270.0 (uL) 270/6=45
- Transfer Master mix("M") to 5 pcr tubes in "M" strip
- Transfer Master mix("MQ") to 5 pcr tubes in "MQ" strip
- Add different primer in each reaction. Adding order: B -> Y -> R-> G-> O from left to right of each strip
- Perform reaction in thermocycler with program "MPCR-52" Tm=52, Amplifying 35 Cycles.