Daniel:Notebook/ComboLock/2017-1-6: Difference between revisions
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>Djacobse (Created page with "=Version 2 Oligos= Back to Calendar The version 2 oligos have several changes. The main is that they are shorter and use a UMI on the padlock i...") |
>Djacobse |
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==Protocol== | ==Protocol-Phase 1== | ||
<ol> | <ol> | ||
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<li>C Probe Hybridization</li> | <li>C Probe Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Combine | <li>Combine following into a 0.2 mL tube</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B1A0C7;font-size:12pt" align="center" | |||
| align="center" width="130" height="30" | Reagent | |||
|style="font-weight:bold" width="67" | uL per Sample | |||
|style="font-weight:bold" width="65" | uL Total | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | PCCA | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | Mirror C Probe B reaction | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Wash buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16 | |||
|- style="background-color:#BFBFBF;font-size:12pt;font-weight:bold" | |||
| height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 40 | |||
|} | |||
<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; '''start part | <li>Incubate at 40C for 1 hour with agitation; '''start part 4 after incubation begins'''</li> | ||
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> | <li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> | ||
<li>Repeat wash step above</li> | <li>Repeat wash step above</li> | ||
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li> | <li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li> | ||
</ol> | </ol> | ||
==Protocol-Phase 2== | |||
<li>Phosphorylation-Lock oligo</li> | <li>Phosphorylation-Lock oligo</li> | ||
<ol type="A"> | <ol type="A"> | ||
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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=" | | width="140" height="30" | Reagent | ||
| width="92" | Stock Conc | | width="92" | Stock Conc | ||
| width="91" | Final Conc./Amount | | width="91" | Final Conc./Amount | ||
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<li>Heat kill enzyme with 15 minutes at 65C</li> | <li>Heat kill enzyme with 15 minutes at 65C</li> | ||
</ol> | </ol> | ||
****** | |||
<li>Lock and Latch Hybridization/Ligation</li> | <li>Lock and Latch Hybridization/Ligation</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up the following master mix</li> | <li>Set up the following master mix; '''Note: for sample 4 add un-phosphorylated Lock0201'''</li> | ||
{| 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:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="150" height="30" | Reagent | ||
| width="65" | Stock Conc | | width="65" | Stock Conc | ||
| width=" | | width="65" | Final Conc./Amt | ||
| width="65" | uL added | | width="65" | uL added | ||
| width="65" | Master Mix ( | | width="65" | Master Mix (6.2X) | ||
| width="65" | Master Mix (2.2X) | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | | height="45" valign="bottom" | T4 Ligase Reaction Buffer | ||
| align="center" | | | align="center" | 5X | ||
| align="center" | 1X | | align="center" | 1X | ||
| align="center" align="center" | | | align="center" align="center" | 4 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 24.8 | ||
| align="center" align="center" valign="bottom" | 8.8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
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| align="center" | 10 pmol | | align="center" | 10 pmol | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 6.2 | ||
| align="center" align="center" valign="bottom" | 2.2 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height=" | | height="45" valign="bottom" | Lock Oligo Phosphate Reaction | ||
| align="center" | (5 uM Lock) | | align="center" | (5 uM Lock) | ||
| align="center" | 10 pmol | | align="center" | 10 pmol | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 12.4 | ||
| align="center" valign="bottom" | *2.2* | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
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| align="center" | NA | | align="center" | NA | ||
| align="center" | NA | | align="center" | NA | ||
| align="center" align="center" | | | align="center" align="center" | 12 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 86.8 | ||
| align="center" align="center" valign="bottom" | 39.2 | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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| align="center" align="center" | | | align="center" align="center" | | ||
|style="font-weight:bold" align="center" align="center" | 19 | |style="font-weight:bold" align="center" align="center" | 19 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 74.4 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 26.4 | |||
|} | |} | ||
<li>Add 19 uL probe mix to each sample; for sample 3 don't add activated | <li>Add 19 uL probe mix to each sample; for sample 3 don't add activated lock, add normal lock0201</li> | ||
<li>Incubate at | <li>Incubate at 65C for 5 min</li> | ||
<li>Lower temperature to 40C; incubate 20 minutes</li> | |||
<li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> | <li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> | ||
<li>Add 1 uL T4 Ligase to each reaction</li> | <li>Add 1 uL T4 Ligase to each reaction</li> | ||
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<li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | ||
<li>Wash twice with 100 uL wash buffer</li></ol> | <li>Wash twice with 100 uL wash buffer</li></ol> | ||
****** | |||
<li>Padlock Hybridization</li> | <li>Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Make following master mix</li> | <li>Make 2 of the following master mix</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>8.2 uL 10 uM padlock0401 or Padlock0401'''A'''</li> | ||
<li> | <li>8.2 uL Amp Ligase 10X reaction buffer</li> | ||
<li> | <li>65.6 uL nfH2O</li></ol> | ||
<li>Resuspend sample in 20 uL padlock buffer</li> | <li>Resuspend sample in 20 uL padlock buffer; incubate A samples with Padlock0401 and B samples with Padlock0401'''A'''</li> | ||
<li>Incubate at 37C for 30 minutes</li></ol> | |||
****** | |||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Prepare | <li>Prepare 8.5X 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="140" height="30" | Reagent | |||
| width="65" | Stock Conc | |||
| width="65" | Final Amount | |||
| width="65" | 1x Vol (uL) | |||
| width="65" | MM Vol (8.5x) (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" | 68 | |||
|- 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" | 5.1 | |||
|- 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" | 25.5 | |||
|- 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" | 17 | |||
|- 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" | 17 | |||
|- 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" | 27.2 | |||
|- 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" | | |||
|style="font-weight:bold" align="center" align="center" | 20 | |||
|style="font-weight:bold" align="center" align="center" | 140 | |||
|} | |||
<li>Add 20 uL phusion mix to 20 uL reaction</li> | |||
<li>Incubate at 40C for 4 hours</li> | |||
</ol> | |||
Continued [[Daniel:Notebook/ComboLock/2017-1-7|tomorrow]] | |||
===Buffers=== | ===Buffers=== | ||
Line 264: | Line 378: | ||
|} | |} | ||
[[Category:ComboLock]] [[Category:20170106]] |
Latest revision as of 19:08, 16 February 2017
Version 2 Oligos[edit]
The version 2 oligos have several changes. The main is that they are shorter and use a UMI on the padlock instead of the latch. I will test these oligos and the new protocol. I'm also going to check
Sample Matrix[edit]
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | No phosphate activation |
Protocol-Phase 1[edit]
- Phosphorylation-MirrorC Probe B (PCCB-02)
- Set up reaction according to table
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- 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 following 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; start part 4 after incubation begins
- 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
- Phosphorylation-Lock oligo
- Set up reaction according to table
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- Lock and Latch Hybridization/Ligation
- Set up the following master mix; Note: for sample 4 add un-phosphorylated Lock0201
- Add 19 uL probe mix to each sample; for sample 3 don't add activated lock, add normal lock0201
- Incubate at 65C for 5 min
- Lower temperature to 40C; incubate 20 minutes
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 30 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
- Padlock Hybridization
- Make 2 of the following master mix
- 8.2 uL 10 uM padlock0401 or Padlock0401A
- 8.2 uL Amp Ligase 10X reaction buffer
- 65.6 uL nfH2O
- Resuspend sample in 20 uL padlock buffer; incubate A samples with Padlock0401 and B samples with Padlock0401A
- Incubate at 37C for 30 minutes
- Circularization
- Prepare 8.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 40C for 4 hours
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Polynucleotide Kinase Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
PCCB-02 | 10 uM | 100 pmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | uL per Sample | uL Total |
PCCA | 1 | 8 |
Mirror C Probe B reaction | 2 | 16 |
Wash buffer | 2 | 16 |
Total | 5 | 40 |
Protocol-Phase 2[edit]
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 DNA Ligase Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
Lock0201 | 10 uM | 10 pmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (6.2X) | Master Mix (2.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 24.8 | 8.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 6.2 | 2.2 |
Lock Oligo Phosphate Reaction | (5 uM Lock) | 10 pmol | 2 | 12.4 | *2.2* |
nfH2O | NA | NA | 12 | 86.8 | 39.2 |
Total | 19 | 74.4 | 26.4 |
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (8.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 68 |
dNTP | 1 mM | 600 pmol | 0.6 | 5.1 |
Betaine | 5 M | 15 umol | 3 | 25.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 17 |
Amp Ligase | 5 U/uL | 10 U | 2 | 17 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 27.2 |
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 |