Daniel:Notebook/ComboLock/2016-6-30: Difference between revisions
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<li>165 uL KAPA SyberFast MM</li> | <li>165 uL KAPA SyberFast MM</li> | ||
<li> | <li>116 uL nfH2O</li> | ||
<li>3.3 uL AmpF 6.4</li></ol> | <li>3.3 uL AmpF 6.4</li></ol> | ||
<li>Add 43 uL Master Mix to each well</li> | <li>Add 43 uL Master Mix to each well</li> |
Revision as of 19:32, 30 June 2016
C Probe SOD1/VIM Test (Started yesterday)
Protocol
- Latch and Padlock Hybridization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 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
- 165 uL KAPA SyberFast MM
- 116 uL nfH2O
- 3.3 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
- Size Selection
- Take 20 uL sample from B2,B3,B5, and B6 into separate tubes
- Add 4 uL 6X loading dye to each tube
- Aliquot 24 uL each into 4 lanes (last one will have 20 uL)
- 5 uL ladder with 1 uL gel loading dye
- Run gel for 25 min at 220V
- Stain with 3 uL SYBR Gold for 3 min
- Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Centrifuge gel at 14000rpm for 1.5 minutes
- Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
- Incubate at 37C for at least an hour with vigorous shaking
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer supernatant to a nanosep column
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer flow through to a 2 mL tube
- Ethanol Precipitation
- Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
- Incubate overnight at -80C; continued tomorrow
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 9 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 10 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 11 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 12 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 13 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | NTC | 14 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |