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

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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| 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"
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| width="82" height="39" | Reagent
| width="120" height="39" | Reagent
| width="65" | Stock
| width="65" | Stock
| width="65" | uL added
| width="65" | uL added
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<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<ol type="A">
<li>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</li>
<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>Heat at 65C for 5 minutes</li>
<li>Chill on ice for 3 minutes</li>
<li>Chill on ice for 3 minutes</li>

Latest revision as of 16:40, 20 October 2016

Padlock0301 Test[edit]

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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