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...")
 
>Djacobse
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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>
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</ol>

Revision as of 19:24, 30 June 2016

C Probe SOD1/VIM Test (Started yesterday)

Back to Calendar

Protocol

  1. Latch and Padlock Hybridization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. 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
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR

Antibody Stage 2 Test 2 (Started 6-28-2016)

  1. Size Selection
    1. Take 20 uL sample from B2,B3,B5, and B6 into separate tubes
    2. Add 4 uL 6X loading dye to each tube
    3. Aliquot 24 uL each into 4 lanes (last one will have 20 uL)
    4. 5 uL ladder with 1 uL gel loading dye
    5. Run gel for 25 min at 220V
    6. Stain with 3 uL SYBR Gold for 3 min
    7. 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
    8. Centrifuge gel at 14000rpm for 1.5 minutes
    9. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
    10. Incubate at 37C for at least an hour with vigorous shaking
    11. Centrifuge at 14000rpm for 1.5 minutes
    12. Transfer supernatant to a nanosep column
    13. Centrifuge at 14000rpm for 1.5 minutes
    14. Transfer flow through to a 2 mL tube
  2. Ethanol Precipitation
    1. Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
    2. Incubate overnight at -80C; continued tomorrow