Daniel:Notebook/ComboLock/2017-1-6: Difference between revisions
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>Djacobse |
>Djacobse |
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==Protocol== | ==Protocol-Phase 1== | ||
<ol> | <ol> | ||
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<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"> | ||
Line 253: | Line 256: | ||
<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="119" 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 | |||
|} | |||
===Buffers=== | ===Buffers=== |
Revision as of 01:19, 6 January 2017
Version 2 Oligos
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
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
- 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 3 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
- Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
- Incubate at 95C for 5 min
- 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 following master mix
- 16.4 uL 10 uM padlock0401
- 16.4 uL Amp Ligase 10X reaction buffer
- 131.2 uL nfH2O
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 30 minutes
- Circularization
- Prepare 8.5X uL Phusion mix
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 |
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
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 (8.2X) |
T4 Ligase Reaction Buffer | 10X | 1X | 2 | 16.4 |
Latch Oligo | 10 uM | 10 pmol | 1 | 8.2 |
Lock Oligo Phosphate Reaction | (5 uM Lock) | 10 pmol | 2 | 16.4 |
nfH2O | NA | NA | 14 | 114.8 |
Total | 19 | 155.8 |
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 |
Buffers
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 |