Daniel:Notebook/ComboLock/2017-1-13: Difference between revisions

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(Created page with "=Version 3 Oligos= Back to Calendar The main difference between version 2 oligos and version 3 oligos i...")
 
>Djacobse
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<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>
<li>Incubate at 40C for 1 hour with agitation; '''start part 4 after incubation begins'''</li>
<li>Incubate at 40C for 1 hour with agitation</li>
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li>
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li>
</ol>
</ol>

Revision as of 19:43, 13 January 2017

Version 3 Oligos

Back to Calendar

The main difference between version 2 oligos and version 3 oligos is only adapter length. The version 3 oligos use the original 18bp adapters instead of the 15bp ones. This helps the annealing temperatures be high, which is better for enzymes such as Amp Ligase and Phusion. The other difference is that the version 3 oligos have phosphate groups on the 5'

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C probes
Sample 4 (AB) No phosphate activation

Protocol

  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
    2. Reagent uL per Sample uL Total
      PCCA-03 1 6
      PCCB-03 1 6
      Wash buffer 3 18
      Total 5 30
    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
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant