Daniel:Notebook/ComboLock/2016-10-5: Difference between revisions

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>Djacobse
>Djacobse
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[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]
===Buffers===
'''Wash/Binding Buffer'''
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Reagent
| width="65" | Stock
| width="65" | Final
| width="65" | Dilution
| width="65" | Amt in 10 mL
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | NaCl
| align="center" valign="bottom" | 1.5 M
| align="center" valign="bottom" | 0.5 M
| align="center" align="center" valign="bottom" | 3
| align="center" valign="bottom" | 3.33 mL
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Tris-HCl
| align="center" valign="bottom" | 500 mM
| align="center" valign="bottom" | 20 mM
| align="center" align="center" valign="bottom" | 25
| align="center" valign="bottom" | 400 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | EDTA
| align="center" valign="bottom" | 0.5M
| align="center" valign="bottom" | 1 mM
| align="center" align="center" valign="bottom" | 500
| align="center" valign="bottom" | 20 uL
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | nf H2O
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 6.25 mL
|}


==Protocol==
==Protocol==
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<ol>
<ol>
<li>Template-Bead Binding</li>
<li>Template-Bead Binding</li>
<ol type="A">
<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>
Line 44: Line 87:
<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>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li>
</ol>
</ol>
<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>

Revision as of 19:56, 5 October 2016

C Probe Positive Control Test

Back to Calendar

Buffers

Wash/Binding Buffer

Reagent Stock Final Dilution Amt in 10 mL
NaCl 1.5 M 0.5 M 3 3.33 mL
Tris-HCl 500 mM 20 mM 25 400 uL
EDTA 0.5M 1 mM 500 20 uL
nf H2O NA NA NA 6.25 mL

Protocol

  C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) Template
Sample 1 X X X
Sample 2 X X  
Sample 3     X
  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 and 3 uL CPH 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 30 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow