Daniel:Notebook/ComboLock/2016-12-13: Difference between revisions
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<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>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> | ||
Line 51: | Line 54: | ||
<li>12.2 uL Amp10X buffer</li> | <li>12.2 uL Amp10X buffer</li> | ||
<li>85.4 uL nfH2O</li></ol> | <li>85.4 uL nfH2O</li></ol> | ||
<li>Incubate at 60C for 1.5 hours</li></ol> | <li>Incubate at 60C for 1.5 hours</li></ol> | ||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare 7X uL Phusion mix</li> | <li>Prepare 7X uL Phusion mix</li> | ||
{| 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="150" height="30" | Reagent | |||
| width="120" | Stock Conc | |||
| width="74" | Final Amount | |||
| width="65" | 1x Vol (uL) | |||
| width="90" | MM Vol (7x) (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | NAD+ | |||
| align="center" | 5 mM | |||
| align="center" | 40 nmol | |||
| align="center" align="center" | 8 | |||
| align="center" align="center" | 56 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | dNTP | |||
| align="center" | 1 mM | |||
| align="center" | 600 pmol | |||
| align="center" align="center" | 0.6 | |||
| align="center" align="center" | 4.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Betaine | |||
| align="center" | 5 M | |||
| align="center" | 15 umol | |||
| align="center" align="center" | 3 | |||
| align="center" align="center" | 21 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | 10X AmpLigase Buffer | |||
| align="center" | 10X | |||
| align="center" | 1X | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 14 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" | 5 U/uL | |||
| align="center" | 10 U | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 14 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | Phusion HF DNA Polymerase | |||
| align="center" | 2000 U/mL | |||
| align="center" | 6.4U | |||
| align="center" align="center" | 3.2 | |||
| align="center" align="center" | 22.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nf H2O | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" align="center" | 1.2 | |||
| align="center" align="center" | 8.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 140 | |||
|} | |||
<li>Add 20 uL Phusion mix to 20 uL of reaction</li> | <li>Add 20 uL Phusion mix to 20 uL of reaction</li> | ||
<li>Incubate at 60C for 2 hours</li></ol> | <li>Incubate at 60C for 2 hours</li></ol> |
Latest revision as of 21:49, 15 December 2016
Mirror Probe Test[edit]
This is a continuation of the failed experiment from Friday with a corrected version of the template.
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probe |
Protocol[edit]
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add following to a fresh 0.2 mL tube
- 12.2 uL Latch0001, 10 uM stock
- 12.2 uL Padlock0201, 10 uM stock
- 12.2 uL Amp10X buffer
- 85.4 uL nfH2O
- Incubate at 60C for 1.5 hours
- Circularization
- Prepare 7X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (7x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 56 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.2 |
Betaine | 5 M | 15 umol | 3 | 21 |
10X AmpLigase Buffer | 10X | 1X | 2 | 14 |
Amp Ligase | 5 U/uL | 10 U | 2 | 14 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 22.4 |
nf H2O | 1.2 | 8.4 | ||
Total | 20 | 140 |
Continued tomorrow
Buffers[edit]
Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 20 mL |
NaCl | 1.5 M | 0.5 M | 3 | 6.66 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 800 uL |
EDTA | 0.5M | 1 mM | 500 | 40 uL |
nf H2O | NA | NA | NA | 12.5 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 |