Daniel:Notebook/ComboLock/2016-8-20: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Positive Control Amplicon Test (Started Thursday)= Back to Calendar ==Protocol== <ol start="6"> <li>S...")
 
>Djacobse
 
(4 intermediate revisions by the same user not shown)
Line 8: Line 8:
<li>Size Selection</li>
<li>Size Selection</li>
<ol type="A">
<ol type="A">
<li>Combine 20 uL sample from each of the following sample lane: 1A, 1B, 2A, 2B, 3A</li>
<li>Add 20 uL sample (lanes 1-4), 5 uL 6X loading dye and 5 uL TBE to sample lanes</li>
<ol type="a">
<li>1A: human, normal, RVC hybridization buffer</li>
<li>2A: human, negative control, RVC hybridization buffer</li>
<li>1B: human, normal, normal hybridization buffer</li>
<li>2B: human, negative control, normal hybridization buffer</li>
<li>3B: mouse, normal, normal hybridization buffer</li>
</ol>
<li>Add 20 uL sample, 5 uL 6X loading dye and 5 uL TBE to sample lanes</li>
<li>Aliquot 30 uL each into 4 sample lanes</li>
<li>Aliquot 30 uL each into 4 sample lanes</li>
<li>2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer</li>
<li>2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer</li>
Line 23: Line 15:
<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>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>
<gallery perrow=2 heights=200px widths=200px>
<gallery perrow=2 heights=200px widths=200px>
File:|Before Image
File:2016-08-20-PosConTest-SizeSelect.png|Before Image
File:|After Image
File:2016-08-20-PosConTest-SizeSelect-After.png|After Image
</gallery>
</gallery>
<li>Centrifuge gel at 14000rpm for 1.5 minutes</li>
<li>Centrifuge gel at 14000rpm for 1.5 minutes</li>
Line 37: Line 29:
<ol type="A">
<ol type="A">
<li>Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample</li>
<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/2016-7-22|tomorrow]]</li></ol>
<li>Incubate overnight at -80C; continued [[Daniel:Notebook/ComboLock/2016-8-22|Monday]]</li></ol>
</ol>
</ol>
[[Category:ComboLock]] [[Category:20160818]]

Latest revision as of 15:59, 22 August 2016

Positive Control Amplicon Test (Started Thursday)[edit]

Back to Calendar

Protocol[edit]

  1. Size Selection
    1. Add 20 uL sample (lanes 1-4), 5 uL 6X loading dye and 5 uL TBE to sample lanes
    2. Aliquot 30 uL each into 4 sample lanes
    3. 2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer
    4. Run gel for 20 min at 250V
    5. Stain with 3 uL SYBR Gold for 3 min
    6. 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
    7. Centrifuge gel at 14000rpm for 1.5 minutes
    8. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
    9. Incubate at 37C for at least an hour with vigorous shaking
    10. Centrifuge at 14000rpm for 1.5 minutes
    11. Transfer supernatant to a nanosep column
    12. Centrifuge at 14000rpm for 1.5 minutes
    13. 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 Monday