Daniel:Notebook/ComboLock/2016-7-2
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C Probe SOD1/VIM Test 2 (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 10 uL exonuclease I (20 units/μL) and 2 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
- VIM-Ab-LPTest2 Results 20160629.png
qPCR Curve
- 2016-07-02-VIM-SOD1-Test2.jpg
Gel Image
- Fix cells
- Pellet cells at 600g for 3 min and resuspend in 5 mL medium with 1.6% paraformaldehyde
- Incubate 10 min at RT
- Wash cells with 5 mL wash buffer
- Pellet cells and permeabilize with ice-cold methanol for 10 min on ice
- Split cells into 1 mL fractions Once in methanol cells may be stored long term
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 | 16 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 17 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 18 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 19 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | NTC | 21 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
qPCR Results[edit]
Human Cell Line Preparation[edit]
The U87MG cells are ready to passage and I'm almost out of frozen cells.