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

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| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 6.25 mL
| align="center" valign="bottom" | 6.25 mL
|}
'''Low Salt 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" | 0.15 M
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 1 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" 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" 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" align="center" valign="bottom" | 8.58 mL
|}
'''KLN Mix'''
{| 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="180" height="31" | Reagent
| width="65" | Stock
| width="65" | uL added
|- style="font-size:12pt"
| height="15"  valign="bottom" | Amp Ligase Buffer
| align="center" valign="bottom" | 10X
| align="center" align="center" valign="bottom" | 1
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | dNTPs
| align="center" valign="bottom" | 100 uM
| align="center" align="center" valign="bottom" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | Hemo Klentaq
| align="center" valign="bottom" | NA
| align="center" align="center" valign="bottom" | 2
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Amp Ligase
| align="center" valign="bottom" | 5 U/uL
| align="center" align="center" valign="bottom" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" valign="bottom" | NA
| align="center" align="center" valign="bottom" | 5


|}
|}
Line 79: Line 153:
<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>
<li>Add 2 uL template oligo per sample to bead solution; incubate at RT for 5 min</li>
<li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li>
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
</ol>
</ol>
<li>C Probe Hybridization</li>
<li>C Probe Hybridization</li>
<ol type="A">
<ol type="A">
<li>Combine 1 uL of each C probe and 3 uL CPH buffer into a 0.2 mL tube</li>
<li>Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube</li>
<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>
Line 106: Line 180:
<li>Circularization</li>
<li>Circularization</li>
<ol type="A">
<ol type="A">
<li>Prepare 30 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/2016-10-6|tomorrow]] </li>
<li>Incubate at 55C overnight; continued [[Daniel:Notebook/ComboLock/2016-10-6|tomorrow]] </li>
</ol></ol>
</ol></ol>
[[Category:ComboLock]] [[Category:20161005]]

Latest revision as of 18:42, 7 October 2016

C Probe Positive Control Test[edit]

Back to Calendar

Buffers[edit]

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

Low Salt Buffer

Reagent Stock Final Dilution Amt in 10 mL
NaCl 1.5 M 0.15 M 10 1 mL
Tris-HCl 500 mM 20 mM 25 400 uL
EDTA 0.5M 1 mM 500 20 uL
nf H2O NA NA NA 8.58 mL

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 1
dNTPs 100 uM 1
Hemo Klentaq NA 2
Amp Ligase 5 U/uL 1
nfH2O NA 5

Protocol[edit]

  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 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
    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