Daniel:Notebook/ComboLock/2017-2-9: Difference between revisions
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>Djacobse (Created page with "=Beads Binding Test= Daniel:Notebook/ComboLock/Back to Calendar Since this should only take a day (now that I have the proper materials), I want to test something: the b...") |
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=Beads Binding Test= | =Beads Binding Test= | ||
[[Daniel:Notebook/ComboLock | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Since this should only take a day (now that I have the proper materials), I want to test something: the beads binding. I want to see at what temp the beads lose their binding ability and at what temp we lose C probes for that matter. To that extent, I have planned the following experiment. It will cover incubation temperatures ranging from RT to 90C and will include a No C probe and no Template negative controls. I will also use positive controls in the qPCR (straight C probes and straight template). | Since this should only take a day (now that I have the proper materials), I want to test something: the beads binding. I want to see at what temp the beads lose their binding ability and at what temp we lose C probes for that matter. To that extent, I have planned the following experiment. It will cover incubation temperatures ranging from RT to 90C and will include a No C probe and no Template negative controls. I will also use positive controls in the qPCR (straight C probes and straight template). | ||
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'''Sample Matrix''' | '''Sample Matrix''' | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="24" | Sample | |||
| width="65" | 1 (AB) | |||
| width="65" | 2 (AB) | |||
| width="65" | 3 (AB) | |||
| width="65" | 4 (AB) | |||
| width="65" | 5 (AB) | |||
| width="65" | 6 (AB) | |||
| width="65" | 7 (AB) | |||
| width="65" | 8 (AB) | |||
| width="65" | 9 (AB) | |||
| width="65" | 10 (AB) | |||
|- style="font-size:12pt" | |||
| height="30" | Condition | |||
| align="center" | RT | |||
| align="center" | No Template | |||
| align="center" | 30C | |||
| align="center" | 40C | |||
| align="center" | 50C | |||
| align="center" | 60C | |||
| align="center" | 70C | |||
| align="center" | 80C | |||
| align="center" | 90C | |||
| align="center" | No C Probe | |||
|} | |||
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<li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> | <li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> | ||
<li>Apply magnet for 30 sec and remove supernatant</li> | <li>Apply magnet for 30 sec and remove supernatant</li> | ||
<li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li> | <li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min; For "No Template" add 2 uL 2 mg/mL BSA-biotin</li> | ||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | ||
</ol> | </ol> | ||
****** | |||
<li>C Probe Hybridization</li> | <li>C Probe Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="180" height="45" | Reagent | ||
| width="65" | uL per Sample | | width="65" | uL per Sample | ||
| width=" | | width="65" | uL Total (Samples+NT) | ||
| width=" | | width="65" | uL Total (No C Probe) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | PCCA-03 (10 uM) | | height="15" valign="bottom" | PCCA-03 (10 uM) | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" | | | align="center" align="center" | 18 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
Line 46: | Line 74: | ||
| height="15" valign="bottom" | PCCB-03 (10 uM) | | height="15" valign="bottom" | PCCB-03 (10 uM) | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" | | | align="center" align="center" | 18 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
Line 53: | Line 80: | ||
| height="15" valign="bottom" | Wash buffer | | height="15" valign="bottom" | Wash buffer | ||
| align="center" align="center" | 3 | | align="center" align="center" | 3 | ||
| align="center" align="center" | | | align="center" align="center" | 54 | ||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
Line 60: | Line 86: | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
| align="center" align="center" | 5 | | align="center" align="center" | 5 | ||
| align="center" align="center" | | | align="center" align="center" | 90 | ||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
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<li>Add 5 uL probe mixture to beads</li> | <li>Add 5 uL probe mixture to beads</li> | ||
<li>Incubate at 40C for 1 hour with agitation</li> | <li>Incubate at 40C for 1 hour with agitation</li> | ||
</ol> | |||
****** | |||
<li>Wash Testing</li> | |||
<ol type="A"> | |||
<li>Repeat following washes 5 times</li> | |||
<ol type="a"> | |||
<li>Add 100 uL wash buffer</li> | |||
<li>Set on magnet for 1 min; '''take 15 uL aside for testing'''</li> | |||
<li>Vortex and incubate for 5 minutes at appropriate incubation temperature</li> | |||
<li>Set on magnet for 1 min; remove supernatant completely</li> | |||
</ol></ol></ol> | |||
Ran out of time; continued [[Daniel:Notebook/ComboLock/2017-2-10|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170209]] |
Latest revision as of 22:41, 13 February 2017
Beads Binding Test[edit]
Since this should only take a day (now that I have the proper materials), I want to test something: the beads binding. I want to see at what temp the beads lose their binding ability and at what temp we lose C probes for that matter. To that extent, I have planned the following experiment. It will cover incubation temperatures ranging from RT to 90C and will include a No C probe and no Template negative controls. I will also use positive controls in the qPCR (straight C probes and straight template).
Sample Matrix and Workflow[edit]
Sample Matrix
Sample | 1 (AB) | 2 (AB) | 3 (AB) | 4 (AB) | 5 (AB) | 6 (AB) | 7 (AB) | 8 (AB) | 9 (AB) | 10 (AB) |
Condition | RT | No Template | 30C | 40C | 50C | 60C | 70C | 80C | 90C | No C Probe |
Workflow Diagram
File:20170208-BeadsTest-Workflow.png
Protocol[edit]
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min; For "No Template" add 2 uL 2 mg/mL BSA-biotin
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine following into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash Testing
- Repeat following washes 5 times
- Add 100 uL wash buffer
- Set on magnet for 1 min; take 15 uL aside for testing
- Vortex and incubate for 5 minutes at appropriate incubation temperature
- Set on magnet for 1 min; remove supernatant completely
Reagent | uL per Sample | uL Total (Samples+NT) | uL Total (No C Probe) |
PCCA-03 (10 uM) | 1 | 18 | 0 |
PCCB-03 (10 uM) | 1 | 18 | 0 |
Wash buffer | 3 | 54 | 10 |
Total | 5 | 90 | 10 |
Ran out of time; continued tomorrow