Daniel:Notebook/ComboLock/2016-9-5: Difference between revisions

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(Created page with "=X3 Dilution Test (Started Monday)= Back to Calendar ==Protocol (from [[Daniel:Notebook/ComboLock/2016-...")
 
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=X3 Dilution Test (Started [[Daniel:Notebook/ComboLock/2016-8-29|Monday]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]


==Protocol (from [[Daniel:Notebook/ComboLock/2016-9-2|yesterday]])==
<ol start="5">
 
<li>Size Select Gel</li>
<ol start="6">
<ol type="A">
<li>For sample lanes
<ol type="a">
<li>Mix 10 uL from each replicate (30 total) in separate tubes for each sample</li>
<li>Use lanes 1A-H and lane A12</li>
<li>Add 6 uL 6X loading dye and 4 uL TBE for 40 uL total</li>
<li>For sample 1, do this twice (80 uL total)</li>
</ol>
<li>For ladder lane mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)</li>
<li>Add 40 uL to each lane</li>
<li>Run gel for 25 minutes at 250V</li>
<li>While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube</li>
<li>Stain gel for 3 minutes with 3 uL SYBR gold</li>
<li>Image in gel doc</li>
<li>Extract desired bands (see gel image) and put into tubes; 2 lanes per tube (2 tubes total)</li>
<li>Image in gel doc post-extraction</li>
<gallery perrow=2 heights=300px widths=300px>
File:|Before Image
File:|After Image
</gallery>
<li>Centrifuge tubes at 14000rpm for 1.5 minutes</li>
<li>Discard 0.5 mL tube and resuspend in 500 uL TE buffer</li>
<li>Incubate at 37C for 1 hour with vigorous shaker</li>
<li>Centrifuge at 14000 rpm for 1.5 minutes</li>
<li>Extract the supernatant and place into NanoSep column</li>
<li>Centrifuge at 14000 rpm for 1.5 minutes</li>
</ol>
<li>Ethanol Precipitation</li>
<li>Ethanol Precipitation</li>
<ol type="A" start="3">
<ol type="A">
<li>Pellet by centrifuging for 25 minutes at 12000rpm at 4C</li>
<li>In a 2 mL tube, add 1250 uL 100% EtOH, 50 uL 3 M NaOAc, and 2 uL glycoblue to 500 uL sample</li>
<li>Remove supernatant and add 750 uL chilled 70% EtOH</li>
<li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2016-9-6|tomorrow]]</li>  
<li>Centrifuge for 12 minutes at 12000rpm at 4C</li>
<li>Remove supernatant and spin in vacuum centrifuge for 10 minutes</li>
<li>Add 20 uL nfH2O and measure in nanodrop</li>
 
===Nanodrop Results===
 
Since these results aren't great, I'm going to try again with size select on [[Daniel:Notebook/ComboLock/2016-9-5|Monday]].
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center"
| width="99" height="36" | Sample
| width="65" | ng/uL
 
|- style="font-size:12pt"  valign="bottom"
| height="15" | Sample A
| align="center" | 2.5
 
|- style="background-color:#D9D9D9;font-size:12pt"  valign="bottom"
| height="15" | Sample B
| align="center" | 5.9


|}


[[Category:Combolock]] [[Category:20160829]]
[[Category:Combolock]] [[Category:20160829]]

Revision as of 22:04, 5 September 2016


  1. Size Select Gel
    1. For sample lanes
      1. Mix 10 uL from each replicate (30 total) in separate tubes for each sample
      2. Use lanes 1A-H and lane A12
      3. Add 6 uL 6X loading dye and 4 uL TBE for 40 uL total
      4. For sample 1, do this twice (80 uL total)
    2. For ladder lane mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
    3. Add 40 uL to each lane
    4. Run gel for 25 minutes at 250V
    5. While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube
    6. Stain gel for 3 minutes with 3 uL SYBR gold
    7. Image in gel doc
    8. Extract desired bands (see gel image) and put into tubes; 2 lanes per tube (2 tubes total)
    9. Image in gel doc post-extraction
    10. Centrifuge tubes at 14000rpm for 1.5 minutes
    11. Discard 0.5 mL tube and resuspend in 500 uL TE buffer
    12. Incubate at 37C for 1 hour with vigorous shaker
    13. Centrifuge at 14000 rpm for 1.5 minutes
    14. Extract the supernatant and place into NanoSep column
    15. Centrifuge at 14000 rpm for 1.5 minutes
  2. Ethanol Precipitation
    1. In a 2 mL tube, add 1250 uL 100% EtOH, 50 uL 3 M NaOAc, and 2 uL glycoblue to 500 uL sample
    2. Incubate at -80C overnight; continued tomorrow