Daniel:Notebook/ComboLock/2017-2-9: Difference between revisions

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(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/Back to Calendar]]
[[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="130" height="30" | Reagent
| width="180" height="45" | Reagent
| width="65" | uL per Sample
| width="65" | uL per Sample
| width="90" | uL Total (Samples+NT)
| width="65" | uL Total (Samples+NT)
| width="90" | uL Total (No Phosphate)
| width="65" | uL Total (No C Probe)
| width="90" | 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" | 4
| align="center" align="center" | 18
| align="center" align="center" | 2
| 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" | 4
| align="center" align="center" | 18
| align="center" | 2***
| 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" | 12
| align="center" align="center" | 54
| align="center" align="center" | 6
| 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" | 20
| align="center" align="center" | 90
| align="center" align="center" | 10
| 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]

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).

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]

  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. 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
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine following into a 0.2 mL tube
    2. 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
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 5 uL probe mixture to beads
    5. Incubate at 40C for 1 hour with agitation
  3. Wash Testing
    1. Repeat following washes 5 times
      1. Add 100 uL wash buffer
      2. Set on magnet for 1 min; take 15 uL aside for testing
      3. Vortex and incubate for 5 minutes at appropriate incubation temperature
      4. Set on magnet for 1 min; remove supernatant completely

Ran out of time; continued tomorrow