Daniel:Notebook/ComboLock/2016-8-23
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Positive Control Amplicon Test 2 (Started yesterday)
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- Master Mix
- 277.5 uL KAPA SyberFast MM
- 194.25 uL nfH2O
- 5.55 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- Phosphorylation
- In a 0.2 mL tube, add ingredients according to table
- Incubate at 37C for 30 min
- Ligation
- Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
- Add amplicons according to following table
- Heat reaction to 95C for 5 min
- Lower temp to 55C
- Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
- Incubate at 55C for 2 hours
- Heat to 95 C to denature dsDNA
- Purify with ssDNA column
- ssDNA Column
- Add 100 uL Binding Buffer to the sample; mix well
- Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
- Add 150 uL 100% EtOH to flow through; mix well
- Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Centrifuge empty column at 14000 rpm for 2 minutes
- Transfer to empty 1.5mL centrifuge tube (low bind)
- Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute
Sample | Name | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | X2-1X | A1 | 33 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | X2-5X | B1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | X2-10X | C1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | X2-50X | D1 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | X3-1X | E1 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | X3-5X | F1 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | X3-10X | G1 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | X3-50X | H1 | 40 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | X2Flip | A12 | 41 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10 | X3Flip | B12 | 42 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | PCR NTC | C12 | 43 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |