Daniel:Notebook/ComboLock/2017-6-15: Difference between revisions
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=Antibody Detection Test-Positive Control (Started [[Daniel:Notebook/ComboLock/2017-6-14|Yesterday]])= | =Antibody Detection Test-Positive Control (Started [[Daniel:Notebook/ComboLock/2017-6-14|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
==Protocol-Part 3== | ==Protocol-Part 3== | ||
Line 98: | Line 98: | ||
<li>Incubate at 95C for 5 minutes</li> | <li>Incubate at 95C for 5 minutes</li> | ||
<li>Hold at 16C</li> | <li>Hold at 16C</li> | ||
</ol></ol> | </ol> | ||
<li>Exonuclease Digestion</li> | |||
<ol type="A"> | |||
<li>Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)</li> | |||
<li>Add 2 uL to each sample, mix by swirling pipette tip</li> | |||
<li>Incubate for 2 hours at 37C</li> | |||
<li>Heat kill by incubating for 5 min at 95C</li> | |||
</ol> | |||
</ol> | |||
==Protocol-Part 4== | |||
<ol start="7"> | |||
<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="210" height="30" | Reagent | |||
| width="95" | Single Rxn (uL) | |||
| width="95" | Master Mix X (10.2X) | |||
|- style="font-size:12pt" | |||
| height="15" | Enzyme | |||
| align="center" | NA | |||
| align="center" | Thermo | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | p12RC*** (10 uM) | |||
| align="center" align="center" valign="bottom" | 0.2 | |||
| align="center" align="center" valign="bottom" | 2.04 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer LLRC*** (10 uM) | |||
| align="center" align="center" valign="bottom" | 0.2 | |||
| align="center" align="center" valign="bottom" | 2.04 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (10 mM) | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5.1 | |||
|- 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" | 20.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- 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" | 4.08 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 11.7 | |||
| align="center" align="center" valign="bottom" | 119.34 | |||
|- 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" | 153 | |||
|} | |||
<li>Add 15 uL master mix to each tube</li> | |||
<li>Add 4 uL sample to appropriate tubes</li> | |||
<li>Heat reactions up to 95C for 5 min</li> | |||
<li>Cool to 55C and incubate for 15 minutes</li> | |||
<li>Cool to 30C and add 1 uL enzyme</li> | |||
<li>Incubate for 3 hours at 30C</li> | |||
<li>Incubate for 65 minutes at 20C to denature</li> | |||
</ol> | |||
<li>Bglii digestion</li> | |||
<ol type="A"> | |||
<li>Make the following reactions; '''Do not add Bglii yet'''</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
|style="background-color:#8DB4E2" width="110" height="30" | Reagent | |||
|style="background-color:#8DB4E2" width="95" | Single Rxn Vol | |||
|style="background-color:#95B3D7" width="95" | Master Mix (10.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RCA Rxn | |||
| align="center" align="center" valign="bottom" | 7 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer 3.1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 20.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Bglii | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 102 | |||
|- style="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" | 122.4 | |||
|} | |||
<li>Aliquot 12 uL master mix into new tube</li> | |||
<li>Add 7 uL appropriate sample</li> | |||
<li>Mix with vortexer, spin down, and add 1 uL Bglii</li> | |||
<li>Incubate 2 hr 37C</li> | |||
<li>Heat kill with 65C for 20 min</li> | |||
<li>Hold at 12C</ol> | |||
</ol> |
Revision as of 18:58, 15 June 2017
Antibody Detection Test-Positive Control (Started Yesterday)
Protocol-Part 3
- Latch and Bolt Hybridization/Ligation
- Heat kill enzyme by incubating at 65C for 10 minutes
- Padlock Hybridization
- Add 20 uL of following 6.2X Master Mix
- 6.2 uL 10 uM Padlock0601
- 12.4 uL 10X Amp Ligase buffer
- 105.4 uL nfH2O
- Incubate at 70C for 5 min
- Incubate at 50C for 15 minutes
- Wash once with wash buffer*Do not wash sample 5
- Resuspend in 20 uL 1X Amp Ligase buffer
- Circularization
- For phusion samples, make the following master mix
- Add 20 uL circularization mix to each sample
- Incubate at 55C for 2 hours
- Incubate at 95C for 5 minutes
- Hold at 16C
- Exonuclease Digestion
- Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 2 hours at 37C
- Heat kill by incubating for 5 min at 95C
******
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (10.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 52 |
dNTP | 1 mM | 600 pmol | 0.6 | 3.9 |
Betaine | 5 M | 15 umol | 3 | 19.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 13 |
Amp Ligase | 5 U/uL | 10 U | 2 | 13 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 20.8 |
nf H2O | 1.2 | 7.8 | ||
Total | 20 | 130 |
Protocol-Part 4
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Add 4 uL sample to appropriate tubes
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- Hold at 12C
Reagent | Single Rxn (uL) | Master Mix X (10.2X) |
Enzyme | NA | Thermo |
Sample | 4 | 0 |
p12RC*** (10 uM) | 0.2 | 2.04 |
RCA Primer LLRC*** (10 uM) | 0.2 | 2.04 |
dNTP (10 mM) | 0.5 | 5.1 |
10X Buffer | 2 | 20.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 4.08 |
nfH2O | 11.7 | 119.34 |
Total | 20 | 153 |
Reagent | Single Rxn Vol | Master Mix (10.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 20.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 102 |
Total | 20 | 122.4 |