Daniel:Notebook/ComboLock/2016-7-6: Difference between revisions
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>Djacobse |
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(9 intermediate revisions by the same user not shown) | |||
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<gallery perrow=2 heights=200px widths=200px> | <gallery perrow=2 heights=200px widths=200px> | ||
File:|qPCR Curve | File:CProbe-VIM-SOD1-20160706-Sequential.png|qPCR Curve | ||
File:|Gel Image | File:2016-07-06-VIM-SOD1-Seq.jpg|Gel Image | ||
</gallery> | </gallery> | ||
=Antibody Stage 2 Test 2 (Started [[Daniel:Notebook/ComboLock/2016- | ==Protocol Part II== | ||
Since lanes 1-5 showed product, I'm going to sequence all of them. This is disheartening as MORE samples showed signal now that I changed to sequential hybridization. | |||
<ol start="5"> | |||
<li>Size Selection</li> | |||
<ol type="A"> | |||
<li>Combine 20 uL sample from each of the five samples</li> | |||
<ol type="a"><li>Add only 10 uL from lane 3 plus 10 uL TBE since lane 3 had greatest signal</li></ol> | |||
<li>Add 30 uL 6X loading dye</li> | |||
<li>Aliquot 30 uL each into 4 lanes</li> | |||
<li>2 uL ladder with 5 uL gel loading dye and 23 uL TBE buffer</li> | |||
<li>Run gel for 45 min at 250V</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> | |||
<gallery perrow=2 heights=200px widths=200px> | |||
File:|Before Image | |||
File:|After Image | |||
</gallery> | |||
<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/2016-7-7|tomorrow]]</li></ol> | |||
</ol> | |||
=Antibody Stage 2 Test 2 (Started [[Daniel:Notebook/ComboLock/2016-6-28|6-28-2016]])= | |||
Continued from results on [[Daniel:Notebook/ComboLock/2016-7-1|7-1-2016]] | Continued from results on [[Daniel:Notebook/ComboLock/2016-7-1|7-1-2016]] | ||
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|} | |} | ||
Sequencing Sample: '''DEJ-CL-P01''' | |||
=C Probe SOD1/VIM Test (Started [[Daniel:Notebook/ComboLock/2016-6-29|6-29-2016]])= | |||
Continued from [[Daniel:Notebook/ComboLock/2016-7-1|7-1]]. I added more human than mouse since I am more interested in the human samples (although it is still important to check the mouse). | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#DDD9C4;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="30" | Sample | |||
| width="65" | ng/uL | |||
| width="65" | uL added | |||
| width="65" | final mass (ng) | |||
|- style="font-size:12pt" | |||
| height="15" | Hs-Sample 2 | |||
| align="center" align="center" | 38.5 | |||
| align="center" align="center" valign="bottom" | 15 | |||
| align="center" align="center" valign="bottom" | 577.5 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" | mm-Sample 4 | |||
| align="center" align="center" | 43.3 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 433 | |||
|} | |||
Sequencing Sample: '''DEJ-CL-C02''' | |||
[[Category:ComboLock]] [[Category:20160705]] [[Category:20160628]] [[Category:20160629]] |
Latest revision as of 22:54, 6 July 2016
Sequential Hybridization Test (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 10 uL exonuclease I (20 units/μL) and 2 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
- Set up reaction according to table
- Master Mix
- 165 uL KAPA SyberFast MM
- 116 uL nfH2O
- 3.3 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- CProbe-VIM-SOD1-20160706-Sequential.png
qPCR Curve
- 2016-07-06-VIM-SOD1-Seq.jpg
Gel Image
- Size Selection
- Combine 20 uL sample from each of the five samples
- Add only 10 uL from lane 3 plus 10 uL TBE since lane 3 had greatest signal
- Add 30 uL 6X loading dye
- Aliquot 30 uL each into 4 lanes
- 2 uL ladder with 5 uL gel loading dye and 23 uL TBE buffer
- Run gel for 45 min at 250V
- 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
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | NTC | 27 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
qPCR Results[edit]
Protocol Part II[edit]
Since lanes 1-5 showed product, I'm going to sequence all of them. This is disheartening as MORE samples showed signal now that I changed to sequential hybridization.
Antibody Stage 2 Test 2 (Started 6-28-2016)[edit]
Continued from results on 7-1-2016
Source | ng/uL | uL added | final mass (ng) |
Ab1 | 15 | 20 | 300 |
Sequencing Sample: DEJ-CL-P01
C Probe SOD1/VIM Test (Started 6-29-2016)[edit]
Continued from 7-1. I added more human than mouse since I am more interested in the human samples (although it is still important to check the mouse).
Sample | ng/uL | uL added | final mass (ng) |
Hs-Sample 2 | 38.5 | 15 | 577.5 |
mm-Sample 4 | 43.3 | 10 | 433 |
Sequencing Sample: DEJ-CL-C02