Daniel:Notebook/ComboLock/2016-7-7: Difference between revisions
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Sample Name: '''DEJ-CL-C03''' | Sample Name: '''DEJ-CL-C03''' | ||
=C Probe/Antibody Mix= | |||
I think it is time to try an experiment with the C-Probes and antibodies. Just to get sequence information, if we can. I think I will stick to the sequential hybridization method for now. | |||
==Buffer Prep== | |||
Prepare 1 mL of hybridization buffer and aliquot in 100 uL fractions. Freeze unused. | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="114" height="30" | Reagent | |||
| width="106" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="65" | Amt in 100 uL (uL) | |||
| width="65" | Amt in 1 mL (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Oligos | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" valign="bottom" | 100 nM | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" valign="bottom" | 1 uL | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Antibodies | |||
| align="center" valign="bottom" | 1500 nM | |||
| align="center" valign="bottom" | 0.5 nM | |||
| align="center" align="center" valign="bottom" | 3000 | |||
| align="center" valign="bottom" | 1 uL | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | BSA | |||
| align="center" valign="bottom" | 50 mg/mL (5%) | |||
| align="center" align="center" valign="bottom" | 0.10% | |||
| align="center" align="center" valign="bottom" | 500 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | RNAsin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" align="center" valign="bottom" | 0.1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | EDTA | |||
| align="center" valign="bottom" | 500 mM | |||
| align="center" valign="bottom" | 5 mM | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 10 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Salmon Sperm DNA | |||
| align="center" valign="bottom" | 10 mg/mL | |||
| align="center" valign="bottom" | 0.1 mg/mL | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" valign="bottom" | 1 uL | |||
| align="center" align="center" valign="bottom" | 10 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" valign="bottom" | 10 uL | |||
| align="center" align="center" valign="bottom" | 100 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 85 uL | |||
| align="center" align="center" valign="bottom" | 877 | |||
|} | |||
==Protocol== | |||
<ol> | |||
<li>Hybridization</li> | |||
<ol type="A"> | |||
<li>Measure cell concentration using 10 uL cell sample in 90 uL nfH2O</li> | |||
<ol type="a"><li>Human- cells/mL</li></ol> | |||
<li>Extract cells to a new tube such that there are 100000 cells present</li> | |||
<ol type="a"><li>Human- uL; Mouse-85 uL</li></ol> | |||
<li>Pellet cells via centrifugation at 600g for 3 min</li> | |||
<li>Add 200 uL Wash Buffer (0.05% Tween 20 in 1X PBS)</li> | |||
<li>Pellet cells via centrifugation at 600g for 3 min</li> | |||
[[Category:ComboLock]] [[Category:20160705]] | [[Category:ComboLock]] [[Category:20160705]] |
Revision as of 18:14, 7 July 2016
Sequential Hybridization Test (Started 7-5-2016)
Protocol
- Ethanol precipitation (from yesterday)
- Centrifuge at 4 C for 25 minutes at 14000 rpm
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge for 15 minutes at minutes at 4 C at 14000 rpm
- Remove supernatant and dry the pellet in the hood
- Resuspend pellet in 20 uL volume nfH20
- Measure in Nanodrop
- Store DNA at 4C
Nanodrop Results
Source | ng/uL |
C Probe-Seq Tube 1 | 243.1 |
C Probe-Seq Tube 2 | 287.9 |
Library Prep
Mixed samples according to table.
Sample | ng/uL | uL added | final mass (ug) |
C Probe-Seq Tube 1 | 243.1 | 15 | 3.6 |
C Probe-Seq Tube 2 | 287.9 | 15 | 4.3 |
Sample Name: DEJ-CL-C03
C Probe/Antibody Mix
I think it is time to try an experiment with the C-Probes and antibodies. Just to get sequence information, if we can. I think I will stick to the sequential hybridization method for now.
Buffer Prep
Prepare 1 mL of hybridization buffer and aliquot in 100 uL fractions. Freeze unused.
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 1 uL | 0 |
Antibodies | 1500 nM | 0.5 nM | 3000 | 1 uL | 0 |
BSA | 50 mg/mL (5%) | 0.10% | 500 | 0.5 | 2 |
RNAsin | 40000 U/mL | 40 U/mL | 1000 | 0.1 | 1 |
EDTA | 500 mM | 5 mM | 100 | 1 | 10 |
Salmon Sperm DNA | 10 mg/mL | 0.1 mg/mL | 100 | 1 uL | 10 |
PBS | 10X | 1X | 10 | 10 uL | 100 |
nf H2O | NA | NA | NA | 85 uL | 877 |
Protocol
- Hybridization
- Measure cell concentration using 10 uL cell sample in 90 uL nfH2O
- Human- cells/mL
- Extract cells to a new tube such that there are 100000 cells present
- Human- uL; Mouse-85 uL
- Pellet cells via centrifugation at 600g for 3 min
- Add 200 uL Wash Buffer (0.05% Tween 20 in 1X PBS)
- Pellet cells via centrifugation at 600g for 3 min