Daniel:Notebook/ComboLock/2016-10-13: Difference between revisions

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<li>Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water</li>
<li>Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water</li>
</ol></ol>
</ol></ol>
===Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center"
| width="65" height="26" | Sample
| width="65" | ng/uL
| width="65" | Volume (uL)
| width="65" | Total (ug)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Experiment
| align="center" align="center" valign="bottom" | 59
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 1.8
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" align="center" valign="bottom" | 25.7
| align="center" align="center" valign="bottom" | 20
| align="center" align="center" valign="bottom" | 0.5
|}


=Padlock0301 Dilution Test=
=Padlock0301 Dilution Test=
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<li>Prepare 10 uL KLN mix</li>
<li>Prepare 10 uL KLN mix</li>
<li>Add 2 uL KLN mix to 20 uL of reaction</li>
<li>Add 2 uL KLN mix to 20 uL of reaction</li>
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-8|tomorrow]] </li>
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-14|tomorrow]] </li>
</ol></ol>
</ol></ol>




[[Category:ComboLock]] [[Category:20161007]] [[Category:20161013]]
[[Category:ComboLock]] [[Category:20161007]] [[Category:20161013]]

Latest revision as of 15:46, 20 October 2016

Padlock0301 Test (Started Friday 10-7)[edit]

Back to Calendar

Library Prep[edit]

  1. Size Select Gel
    1. Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
    2. Extract supernatant and add to nanosep column
    3. Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
  2. Ethanol Precipitation
    1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
    2. Incubate at -80C for a while
    3. Centrifuge for 25 minutes at 12000 rpm at 4C
    4. Remove supernatant and add 750 uL 70% EtOH
    5. Centrifuge for 12 minutes at 12000 rpm at 4C
    6. Remove supernatant and spin in vacuum centrifuge for 10 minutes
    7. Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water

Results[edit]

Sample ng/uL Volume (uL) Total (ug)
Experiment 59 30 1.8
NTC 25.7 20 0.5

Padlock0301 Dilution Test[edit]

Stock Dilutions[edit]

Create the following dilutions of PCC Template by taking 10 uL stock and diluting with 90 uL nfH2O (note:zero is normal 10 uM stock). #6 is the highest concentration, made on Friday 10-7.

Tube No Dilution Molecules Available Amount
6 1.00E+06 1.00E+06 10 amol
7 1.00E+07 1.00E+05 1 amol
8 1.00E+08 1.00E+04 100 zmol
9 1.00E+09 1000 10 zmol
10 1.00E+10 100 1 zmol
11 1.00E+11 10 100 ymol
12 1.00E+12 1 10 ymol

Buffers[edit]

Experiment 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 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
    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. Latch and Padlock Hybridization
    1. Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
    2. Heat at 65C for 5 minutes
    3. Chill on ice for 3 minutes
    4. Add sample to prepared strep beads; vortex to suspend
    5. Incubate for 30 min at 37C with intermittent mixing
    6. Apply magnet and remove supernatant
    7. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    8. Repeat wash step above
    9. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    10. Resuspend with 20 uL 1x Amp Ligase buffer
  4. Circularization
    1. Prepare 10 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow