Daniel:Notebook/ComboLock/2016-11-29: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
Line 45: Line 45:
<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</li>
<li>Incubate at 40C for 1 hour with agitation; '''start part 3 after incubation begins'''</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>

Revision as of 18:00, 29 November 2016

Extended Latch Test

Back to Calendar

The purpose of this experiment is to try and get the circular product. We've had some difficulty with the product containing more of the C-probe (see Nov 18 and Nov 21 results) sequence than they should. So I'm going to try an extended latch that covers the C1 barcode region besides the adapters and UMI. I can give the padlock a 5' phosphate group and then add only Amp ligase. This attach the latch to the padlock at locations where both are present. I can then gap fill.

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C Probe
Sample 4 (AB) No Kinase

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 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
    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; start part 3 after incubation begins
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
  3. Phosphorylation
    1. Set up reaction according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      AmpLigase Reaction Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      Padlock0201 15 uM 150 pmol total 15
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 0
      Total     20
    3. Incubate at 37C for 30 min
  4. Latch and Padlock Hybridization
    1. Add following to a fresh 0.2 mL tube
      1. 12.2 uL LatchS002, 10 uM stock
      2. 12.2 uL Padlock0201, 10 uM stock
      3. 12.2 uL Amp10X buffer
      4. 85.4 uL nfH2O
    2. Add 20 uL probe mix to each sample; incubate 20C for 4 min
    3. Incubate at 95C for 5 min
    4. Incubate at 60C for 1.5 hours
  5. Circularization
    1. Prepare 7X uL Phusion mix
    2. Add 20 uL Phusion mix to 20 uL of reaction
    3. Incubate at 60C for 2 hours