Daniel:Notebook/ComboLock/2017-3-13: Difference between revisions

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>Djacobse
(Created page with "=Bolt Test (Started Wednesday March 8)= Back to Calendar ==Protocol== '''Sample Matrix''' {| class="wi...")
 
>Djacobse
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<ol type="A">
<ol type="A">
<li>Combine following into a 0.2 mL tube</li>
<li>Combine following into a 0.2 mL tube</li>
 
<ol type="a">
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
<li>4 uL each PCCA-03 and PCCB-03 (10 uM each)</li>
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
<li>12 uL wash buffer</li></ol>
| width="200" height="30" | Reagent
| width="85" | uL per Sample
| width="85" | Exp uL Total
| width="85" | No C uL Total
| width="85" | NP uL Total
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | PCCA-03 (10 uM)
| align="center" align="center" | 1
| align="center" align="center" | 4
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 2
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | PCCB-03 (10 uM)
| align="center" align="center" | 1
| align="center" align="center" | 4
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Mirror BC-v2 (10 uM)
| align="center" align="center" | 1
| align="center" align="center" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 2
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Wash buffer
| align="center" align="center" | 3
| align="center" align="center" | 12
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 6
 
|- style="font-size:12pt;font-weight:bold"
| height="15"  valign="bottom" | Total
| align="center" align="center" | 5
| align="center" align="center" | 20
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
 
|}
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add 5 uL probe mixture to beads</li>
<li>Add 5 uL probe mixture to beads</li>

Revision as of 15:57, 13 March 2017

Bolt Test (Started Wednesday March 8)

Back to Calendar

Protocol

Sample Matrix

Sample Condition
Sample 5 (AB) No Ligase
Sample 6 (AB) Normal
  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
    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
      1. 4 uL each PCCA-03 and PCCB-03 (10 uM each)
      2. 12 uL wash buffer
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add 5 uL probe mixture to beads
    4. Incubate at 40C for 1 hour with agitation
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Repeat wash step above