Daniel:Notebook/ComboLock/2016-10-7: Difference between revisions

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==Protocol==
==Protocol==


===Stock Dilutions===
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)
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center"
| width="65" height="45" | Tube No
| width="65" | Dilution
| width="65" | Molecules Available
| width="65" | Amount
|- style="font-size:12pt" align="center"
| align="center" height="15" | 0
| align="center" | 1
| align="center" | 1.00E+12
| 10 pmol
|- style="background-color:#BFBFBF;font-size:12pt" align="center"
| align="center" height="15" | 1
| align="center" | 10
| align="center" | 1.00E+11
| 1 pmol
|- style="font-size:12pt" align="center"
| align="center" height="15" | 2
| align="center" | 100
| align="center" | 1.00E+10
| 100 fmol
|- style="background-color:#BFBFBF;font-size:12pt" align="center"
| align="center" height="15" | 3
| align="center" | 1000
| align="center" | 1.00E+09
| 10 fmol
|- style="font-size:12pt" align="center"
| align="center" height="15" | 4
| align="center" | 10000
| align="center" | 1.00E+08
| 1 fmol
|- style="background-color:#BFBFBF;font-size:12pt" align="center"
| align="center" height="15" | 5
| align="center" | 1.00E+05
| align="center" | 1.00E+07
| 100 amol
|- style="font-size:12pt" align="center"
| align="center" height="15" | 6
| align="center" | 100000
| align="center" | 1.00E+06
| 10 amol
|}
===Samples===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
| width="130" height="45" | Sample
| width="65" | C Probes-VIM (C1+C2)
| width="65" | C Probes-SOD1 (C1+C2)
| width="65" | Template
| width="65" | Dilution
| width="65" | Dilution Amount
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1 (ABC)
| align="center" | X
| align="center" | X
| align="center" | X
| align="center" align="center" valign="bottom" | 0
| align="center" | 10 pmol
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 2
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | None
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 3
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" valign="bottom" | 0
| align="center" valign="bottom" | 10 pmol
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 4 (ABC)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | 2
| align="center" valign="bottom" | 100 fmol
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 5 (ABC)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | 4
| align="center" valign="bottom" | 1 fmol
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 6 (ABC)
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | 6
| align="center" valign="bottom" | 10 amol
|}
===Buffers===
{| 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="120" height="39" | Reagent
| width="65" | Stock
| width="65" | uL added
|- style="font-size:12pt"
| height="30"  valign="bottom" | Amp Ligase Buffer
| align="center" valign="bottom" | 10X
| align="center" align="center" valign="bottom" | 3
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | dNTPs
| align="center" valign="bottom" | 100 uM
| align="center" align="center" valign="bottom" | 3
|- style="font-size:12pt"
| height="15"  valign="bottom" | Hemo Klentaq
| align="center" valign="bottom" | NA
| align="center" align="center" valign="bottom" | 6
|- 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" | 3
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" valign="bottom" | NA
| align="center" align="center" valign="bottom" | 15
|}
===Experiment Protocol===
<ol>
<li>Template-Bead Binding</li>
<ol type="A">
<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>Add 2 uL 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>
</ol>
<li>C Probe Hybridization</li>
<ol type="A">
<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>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>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<li>Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0301 to each sample to a fresh 0.2 mL tube</li>
<li>Heat at 65C for 5 minutes</li>
<li>Chill on ice for 3 minutes</li>
<li>Add sample to prepared strep beads; vortex to suspend</li>
<li>Incubate for 30 min at 37C with intermittent mixing</li>
<li>Apply magnet and remove supernatant</li>
<li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol>
<li>Circularization</li>
<ol type="A">
<li>Prepare 10 uL KLN mix</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>
</ol></ol>


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

Latest revision as of 16:40, 20 October 2016

Padlock0301 Test[edit]

Back to Calendar

This is a test of the padlock0301, which replaces the AmpF/LinkerR region of the padlock with a custom primer set. This is because my experiments on 9-29, 9-30, 10-3, and 10-4, which test the v1 and v2 padlock systems using AmpF and AmpR individually. AmpF has proved particularly dirty so I have designed v3 of the padlocks without it.

Protocol[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)

Tube No Dilution Molecules Available Amount
0 1 1.00E+12 10 pmol
1 10 1.00E+11 1 pmol
2 100 1.00E+10 100 fmol
3 1000 1.00E+09 10 fmol
4 10000 1.00E+08 1 fmol
5 1.00E+05 1.00E+07 100 amol
6 100000 1.00E+06 10 amol

Samples[edit]

Sample C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) Template Dilution Dilution Amount
Sample 1 (ABC) X X X 0 10 pmol
Sample 2 X X   NA None
Sample 3     X 0 10 pmol
Sample 4 (ABC) X X X 2 100 fmol
Sample 5 (ABC) X X X 4 1 fmol
Sample 6 (ABC) X X X 6 10 amol

Buffers[edit]

Reagent Stock uL added
Amp Ligase Buffer 10X 3
dNTPs 100 uM 3
Hemo Klentaq NA 6
Amp Ligase 5 U/uL 3
nfH2O NA 15

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 Padlock0301 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