Daniel:Notebook/ComboLock/2017-1-27: Difference between revisions
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>Djacobse |
>Djacobse |
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(10 intermediate revisions by the same user not shown) | |||
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<li>Add 20 uL phusion mix to 20 uL reaction</li> | <li>Add 20 uL phusion mix to 20 uL reaction</li> | ||
<li>Incubate at 55C for | <li>Incubate at 55C for 2 hours</li> | ||
</ol></ol> | |||
==Protocol-Part3== | |||
<ol start="6"> | |||
<li>Exonuclease Digestion</li> | |||
<ol type="A"> | |||
<li>Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)</li> | |||
<li>Add 2 uL to each sample, mix by swirling pipette tip</li> | |||
<li>Incubate for 1 hour at 37C</li> | |||
<li>Heat kill by incubating for 5 min at 95C</li> | |||
</ol> | </ol> | ||
<li>Rolling Circle Amplification</li> | |||
<ol type="A"> | |||
<li>Prepare master mix according to table below</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="180" height="30" | Reagent | |||
| width="85" | uL Added | |||
| width="90" | Master Mix (16.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 40.5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 32.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 32.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | BSA (10 mg/mL) | |||
| align="center" align="center" valign="bottom" | 0.4 | |||
| align="center" align="center" valign="bottom" | 6.48 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 8.1 | |||
| align="center" align="center" valign="bottom" | 131.22 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 243 | |||
|} | |||
<li>Add 14 uL master mix to each tube</li> | |||
<li>Add sample and to new reaction tubes according to following table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |||
| width="55" height="33" | Sample Code | |||
| width="85" | Phi29 Source | |||
| width="85" | Incubation Time (hrs) | |||
| width="80" | Sample (1AB) | |||
| width="100" | No Template (2AB) | |||
| width="110" | No C Probes (3AB) | |||
| width="60" | No PO4 (4AB) | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | X | |||
| NEB | |||
| 3 hrs | |||
|style="font-weight:bold" | X | |||
|style="font-weight:bold" | X | |||
|style="font-weight:bold" align="center" | | |||
|style="font-weight:bold" align="center" | | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Y | |||
| NEB | |||
| 6 hrs | |||
|style="font-weight:bold" | X | |||
|style="font-weight:bold" align="center" | | |||
|style="font-weight:bold" | X | |||
|style="font-weight:bold" align="center" | | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Z | |||
| NEB | |||
| 12 hrs | |||
|style="font-weight:bold" | X | |||
|style="font-weight:bold" align="center" | | |||
|style="font-weight:bold" align="center" | | |||
|style="font-weight:bold" | X | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Q | |||
| Epicentre | |||
| 3 hrs | |||
|style="font-weight:bold" | X | |||
|style="font-weight:bold" align="center" | | |||
|style="font-weight:bold" align="center" | | |||
|style="font-weight:bold" | X | |||
|} | |||
<li>Incubate at 30C for X hours (see table above)</li> | |||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | |||
Continued [[Daniel:Notebook/ComboLock/2017-1-28|tomorrow]] | Continued [[Daniel:Notebook/ComboLock/2017-1-28|tomorrow]] | ||
[[Category:ComboLock]] [[Category:20170127]] | [[Category:ComboLock]] [[Category:20170127]] |
Latest revision as of 23:46, 27 January 2017
RCA Timing Test (v3 Oligos)[edit]
I'm going to test the v3 oligos again, this time using longer RCA times than the other ones. This is from what I saw in the last experiment, which suggests to me that increased RCA time can potentially yield results. As such, I'll try several times to test the theory.
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 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
- 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
Reagent | uL per Sample | uL Total (Samples+NT) | uL Total (No Phosphate) | uL Total (No C Probe) |
PCCA-03 (10 uM) | 1 | 4 | 2 | 0 |
PCCB-03 (10 uM) | 1 | 4 | 2*** | 0 |
Wash buffer | 3 | 12 | 6 | 10 |
Total | 5 | 20 | 10 | 10 |
Protocol-Phase 2[edit]
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 65C for 5 min; Note: changed from 95C due to this
- Lower temperature to 55C; 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 the following 8.1X master mix (1X)
- 16.2 uL 10 uM padlock0501(2uL)
- 16.2 uL Amp Ligase 10X reaction buffer(2uL)
- 129.6 uL nfH2O (16uL)
- Heat mixture to 95C for 5 minutes; Cooldown on ice
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 30 minutes
- Circularization
- Prepare 8.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 55C for 2 hours
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (8.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 32.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 8.2 |
Lock Oligo | 10 uM | 10 pmol | 1 | 8.2 |
nfH2O | NA | NA | 13 | 106.6 |
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 |
Protocol-Part3[edit]
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 14 uL master mix to each tube
- Add sample and to new reaction tubes according to following table
- Incubate at 30C for X hours (see table above)
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (16.2X) |
Template | 4 | NA |
RCA Primer (10 uM) | 2.5 | 40.5 |
dNTP (1 mM) | 2 | 32.4 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | NA |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 8.1 | 131.22 |
Total | 20 | 243 |
Sample Code | Phi29 Source | Incubation Time (hrs) | Sample (1AB) | No Template (2AB) | No C Probes (3AB) | No PO4 (4AB) |
X | NEB | 3 hrs | X | X | ||
Y | NEB | 6 hrs | X | X | ||
Z | NEB | 12 hrs | X | X | ||
Q | Epicentre | 3 hrs | X | X |
Continued tomorrow