Daniel:Notebook/ComboLock/2016-6-30: Difference between revisions
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>Djacobse (Created page with "=C Probe SOD1/VIM Test (Started yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Latch and...") |
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==Antibody Stage 2 Test 2 (Started [[Daniel:Notebook/ComboLock/2016-6-28#Trial 2|6-28-2016]])== | |||
<ol start="5"> | |||
<li>Size Selection</li> | |||
<ol type="A"> | |||
<li>Take 20 uL sample from B2,B3,B5, and B6 into separate tubes</li> | |||
<li>Add 4 uL 6X loading dye to each tube</li> | |||
<li>Aliquot 24 uL each into 4 lanes (last one will have 20 uL)</li> | |||
<li>5 uL ladder with 1 uL gel loading dye</li> | |||
<li>Run gel for 25 min at 220V</li> | |||
<li>Stain with 3 uL SYBR Gold for 3 min</li> | |||
<li>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</li> | |||
<li>Centrifuge gel at 14000rpm for 1.5 minutes</li> | |||
<li>Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel</li> | |||
<li>Incubate at 37C for at least an hour with vigorous shaking</li> | |||
<li>Centrifuge at 14000rpm for 1.5 minutes</li> | |||
<li>Transfer supernatant to a nanosep column</li> | |||
<li>Centrifuge at 14000rpm for 1.5 minutes</li> | |||
<li>Transfer flow through to a 2 mL tube</li> | |||
</ol> | |||
<li>Ethanol Precipitation</li> | |||
<ol type="A"> | |||
<li>Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample</li> | |||
<li>Incubate overnight at -80C; continued [[Daniel:Notebook/ComboLock/2016-6-29|tomorrow]]</li></ol> | |||
</ol> | </ol> |
Revision as of 19:24, 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
Antibody Stage 2 Test 2 (Started 6-28-2016)
- 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