Daniel:Notebook/ComboLock/2016-8-17: Difference between revisions
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>Djacobse |
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<li>Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube</li> | <li>Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube</li> | ||
<gallery perrow=2 heights=200px widths=200px> | <gallery perrow=2 heights=200px widths=200px> | ||
File:| | File:2016-08-17-PosControlAmplicon-SizeSelect.png|Before Image | ||
File:| | File:2016-08-17-PosControlAmplicon-SizeSelect-After.png|After Image | ||
</gallery> | </gallery> | ||
<li>Shred the gel by centrifuging at 14000rpm for 1 min 30 sec</li> | <li>Shred the gel by centrifuging at 14000rpm for 1 min 30 sec</li> | ||
<li>Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking</li> | <li>Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking</li> | ||
<li>Centrifuge at 12000rpm for 1.5 min</li> | |||
<li>Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm</li> | |||
</ol> | |||
<li>Ethanol Precipitation</li> | |||
<ol type="A"> | |||
<li>Move flow through to a 2 mL tube</li> | |||
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue</li> | |||
<li>Incubate at -80C for 4 hours</li> | |||
<li>Pellet by centrifuging for 25 min at 12000rpm at 4C</li> | |||
<li>Remove supernatant and add 750 uL chilled 70% EtOH</li> | |||
<li>Centrifuge for 12 min at 12000 rpm and 4 C</li> | |||
<li>Remove supernatant and air dry</li> | |||
<li>Resuspend in 30 uL nfH2O and measure in nanodrop</li> | |||
</ol> | |||
===Nanodrop Results=== | |||
{| 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="137" height="28" | Sample | |||
| width="69" | ng/uL ssDNA | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | PCAmp1 (C1-C2) | |||
| align="center" align="center" valign="bottom" | 18.1 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | PCAmp3 (C3-C2) | |||
| align="center" align="center" valign="bottom" | 16.0 | |||
|} | |||
[[Category:ComboLock]] [[Category:20160805]] | [[Category:ComboLock]] [[Category:20160805]] |
Latest revision as of 15:16, 18 August 2016
Positive Control Amplicon Production (Started yesterday)[edit]
Protocol[edit]
- Size select gel
- Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
- Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
- Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
- Run the gel at 250V for 25 min
- Stain with 3 uL SYBR gold for 3 min
- Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
- 2016-08-17-PosControlAmplicon-SizeSelect.png
Before Image
- 2016-08-17-PosControlAmplicon-SizeSelect-After.png
After Image
- Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
- Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
- Centrifuge at 12000rpm for 1.5 min
- Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
- Ethanol Precipitation
- Move flow through to a 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
- Incubate at -80C for 4 hours
- Pellet by centrifuging for 25 min at 12000rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 12 min at 12000 rpm and 4 C
- Remove supernatant and air dry
- Resuspend in 30 uL nfH2O and measure in nanodrop
Nanodrop Results[edit]
Sample | ng/uL ssDNA |
PCAmp1 (C1-C2) | 18.1 |
PCAmp3 (C3-C2) | 16.0 |