Daniel:Notebook/ComboLock/2017-3-16: Difference between revisions
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=Circularization Comparison (T4 Poly/Ligase)= | =Circularization Comparison (T4 Poly/Ligase; Started [[Daniel:Notebook/ComboLock/2017-3-15|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Line 8: | Line 8: | ||
<li>Exonuclease Digestion</li> | <li>Exonuclease Digestion</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)</li> | ||
<li>Add 2 uL to each sample, mix by swirling pipette tip</li> | <li>Add 2 uL to each sample, mix by swirling pipette tip</li> | ||
<li>Incubate for 1.5 hours at 37C</li> | <li>Incubate for 1.5 hours at 37C</li> | ||
Line 78: | Line 78: | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol> | </ol></ol> | ||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 200 uL TBE and 50 uL 6X dye</li> | |||
<li>Aliquot 10 uL per sample on to parafilm</li> | |||
<li>Add 2 uL sample or 1.5 uL ladder to aliquots</li> | |||
<li>Mix and add 10 uL per lane to gel</li> | |||
<li>Run gel at 230V for 24 minutes</li> | |||
<li>Add 2 uL SYBR gold and incubate on shaker for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li></ol> | |||
</ol> | |||
===Pre-RCA Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20170316-qPCR-T4Circ-PreRCA.png|qPCR curves | |||
File:2017-03-16-T4Circ-PreRCA.png|Gel image | |||
</gallery> | |||
==Protocol-Part 3-Y Samples== | |||
<ol start="5"> | |||
<li>Padlock Hybridization-Y Samples</li> | |||
<ol type="A"> | |||
<li>Bake samples at 95 C and vortex to resuspend</li> | |||
<li>Make the following master mix</li> | |||
<ol type="a"> | |||
<li>4.2 uL Padlock0601 (10 uM)</li> | |||
<li>8.4 uL Amp Ligase 10X buffer</li> | |||
<li>50.4 uL nfH2O</li></ol> | |||
<li>Add 15 uL master mix to appropriate tubes</li> | |||
<li>Add 5 uL sample according to [[Daniel:Notebook/ComboLock/2017-3-15#Protocol-Part 3|yesterday's tables]]</li> | |||
<li>Incubate at 50C for 20 minutes</li></ol> | |||
****** | |||
<li>Circularization</li> | |||
<ol type="A"> | |||
<li>Make the following master 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="200" height="30" | Reagent | |||
| width="85" | Stock Conc | |||
| width="85" | Final Amount | |||
| width="85" | 1x Vol (uL) | |||
| width="85" | MM Vol (4.5x) (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" | 36 | |||
|- 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" | 2.7 | |||
|- 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" | 13.5 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X AmpLigase Buffer | |||
| align="center" | 10X | |||
| align="center" | 1X | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 9 | |||
|- 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" | 9 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" 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" | 14.4 | |||
|- 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" | 5.4 | |||
|- 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" | 90 | |||
|} | |||
<li>Follow the workflow in [[Daniel:Notebook/ComboLock/2017-3-15#Protocol-Part 3|yesterday]]'s diagrams</li></ol> | |||
****** | |||
<li>Exonuclease Digestion</li> | |||
<ol type="A"> | |||
<li>Mix 7.5 uL Exo I (20U/uL) and 1.5 uL ExoIII (100U/uL)</li> | |||
<li>Add 2 uL to each sample, mix by swirling pipette tip</li> | |||
<li>Incubate for 1.5 hours at 37C</li> | |||
<li>Heat kill by incubating for 5 min at 95C</li> | |||
</ol></ol> | |||
==Protocol-Part 5== | |||
<ol start="10"> | |||
<li>Column Purification-Qiaquick-Do this for 20 uL from Y and Z samples</li> | |||
<ol type="A"> | |||
<li>Add 100 uL (5X) PB (binding buffer) to 20 uL sample</li> | |||
<li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> | |||
<li>Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;</li> | |||
<li>Repeat wash step;</li> | |||
<li>Dry spin column for 1 minute at 14000 rpm; discard flow through</li> | |||
<li>Let stand with cover open in fume hood for ~5 minutes</li> | |||
<li>Transfer column to a new 1.5 mL eppendorf tube</li> | |||
<li>Elute with 30 uL EB</li> | |||
<li>Let stand 1 minute</li> | |||
<li>Spin for 1 minute at 14000 rpm</li></ol> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#808000;font-size:12pt" align="center" | |||
| width="95" height="30" | Sample Origin | |||
| width="95" | Sample New Letter | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Y | |||
| P | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Z | |||
| Q | |||
|} | |||
****** | |||
<li>Rolling Circle Amplification</li> | <li>Rolling Circle Amplification</li> | ||
'''Sample Conversion Table''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#808000;font-size:12pt" align="center" | |||
| width="95" height="30" | Sample Origin | |||
| width="95" | Sample New Letter | |||
| width="95" | Sample Numbers | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | X | |||
| F | |||
|1-5 | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Y | |||
| G | |||
|1,4 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Z | |||
| H | |||
|1,4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | |||
| height="15" | P | |||
| I | |||
|1,4 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Q | |||
| K | |||
|1,4 | |||
|} | |||
<ol type="A"> | <ol type="A"> | ||
<li> | <li>Prepare 8.2X Master mix according to table below</li> | ||
<li>Add 12.5 uL master mix to each tube</li> | |||
<li>Add 4 uL template according to table</li> | |||
<li>Add 2.5 uL correct primer according to table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="250" height="30" | Reagent | |||
| width="85" | Single Rxn Vol | |||
| width="85" | 13.2X (A) MM uL | |||
| width="85" | 13.2X (B) MM uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- 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" | 33 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RCA Phosphothiorate Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 33 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 66 | |||
| align="center" align="center" valign="bottom" | 66 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 26.4 | |||
| align="center" align="center" valign="bottom" | 26.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 13.2 | |||
| align="center" align="center" valign="bottom" | 13.2 | |||
|- style="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" | 5.28 | |||
| align="center" align="center" valign="bottom" | 5.28 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 5.1 | |||
| align="center" align="center" valign="bottom" | 67.32 | |||
| align="center" align="center" valign="bottom" | 67.32 | |||
|- 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" | 211.2 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 211.2 | |||
|} | |||
<li>Incubate at 95 C for 5 minutes</li> | |||
<li>Lower to 55C for 15 minutes</li> | |||
<li>Lower to 30C for at least 2 minutes</li> | |||
<li>Add 1 uL Phi29 polymerase to each sample</li> | |||
<li>Incubate 3 hours at 30C</li> | |||
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | |||
</ol></ol> | |||
Continued [[Daniel:Notebook/ComboLock/2017-3-17|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170315]] |
Latest revision as of 23:10, 21 March 2017
Circularization Comparison (T4 Poly/Ligase; Started Yesterday)[edit]
Protocol-Part 4[edit]
- Exonuclease Digestion
- Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- qPCR
- Make qPCR master mixes according to following recipe
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170316-T4Circ-PreRCATest.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 200 uL TBE and 50 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn | MMProd (15.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | primer12*/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 15.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 15.2 | 4.2 |
nfH2O | 21 | 319.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 380 | 105 |
Total | 48 | 729.6 | 201.6 |
Pre-RCA Results[edit]
- 20170316-qPCR-T4Circ-PreRCA.png
qPCR curves
- 2017-03-16-T4Circ-PreRCA.png
Gel image
Protocol-Part 3-Y Samples[edit]
- Padlock Hybridization-Y Samples
- Bake samples at 95 C and vortex to resuspend
- Make the following master mix
- 4.2 uL Padlock0601 (10 uM)
- 8.4 uL Amp Ligase 10X buffer
- 50.4 uL nfH2O
- Add 15 uL master mix to appropriate tubes
- Add 5 uL sample according to yesterday's tables
- Incubate at 50C for 20 minutes
- Circularization
- Make the following master mix
- Follow the workflow in yesterday's diagrams
- Exonuclease Digestion
- Mix 7.5 uL Exo I (20U/uL) and 1.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (4.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 36 |
dNTP | 1 mM | 600 pmol | 0.6 | 2.7 |
Betaine | 5 M | 15 umol | 3 | 13.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 9 |
Amp Ligase | 5 U/uL | 10 U | 2 | 9 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 14.4 |
nf H2O | 1.2 | 5.4 | ||
Total | 20 | 90 |
******
Protocol-Part 5[edit]
- Column Purification-Qiaquick-Do this for 20 uL from Y and Z samples
- Add 100 uL (5X) PB (binding buffer) to 20 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
- Repeat wash step;
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Elute with 30 uL EB
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification Sample Conversion Table
- Prepare 8.2X Master mix according to table below
- Add 12.5 uL master mix to each tube
- Add 4 uL template according to table
- Add 2.5 uL correct primer according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 3 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Sample Origin | Sample New Letter |
Y | P |
Z | Q |
******
Sample Origin | Sample New Letter | Sample Numbers |
X | F | 1-5 |
Y | G | 1,4 |
Z | H | 1,4 |
P | I | 1,4 |
Q | K | 1,4 |
Reagent | Single Rxn Vol | 13.2X (A) MM uL | 13.2X (B) MM uL |
Template | 4 | 0 | 0 |
RCA Primer (10 uM) | 2.5 | 33 | 0 |
RCA Phosphothiorate Primer (10 uM) | 2.5 | 0 | 33 |
dNTP (1 mM) | 5 | 66 | 66 |
10X Buffer | 2 | 26.4 | 26.4 |
Phi29 | 1 | 13.2 | 13.2 |
BSA (10 mg/mL) | 0.4 | 5.28 | 5.28 |
nfH2O | 5.1 | 67.32 | 67.32 |
Total | 20 | 211.2 | 211.2 |
Continued tomorrow