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>Djacobse (Created page with "=RCA + Circularization Test= Back to Calendar Repeating the circularization and RCA test using the three different polymerase enzymes to classi...") |
>Djacobse |
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Repeating the circularization and RCA test using the three different polymerase enzymes to classify which is best to use. This will use the C4-C2 template, which is the simplified template that is the equivalent of the two C probe arms ligated and gap-filled. | Repeating the circularization and RCA test using the three different polymerase enzymes to classify which is best to use. This will use the C4-C2 template, which is the simplified template that is the equivalent of the two C probe arms ligated and gap-filled. | ||
==Protocol== | ==Protocol-Part 1== | ||
'''Sample Matrix''' | |||
{| 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="120" height="28" | Sample | |||
| width="190" | Condition | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 (ABC) | |||
| align="center" valign="bottom" | Sample | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 (ABC) | |||
| align="center" valign="bottom" | No Padlock PO4 | |||
|} | |||
<ol> | <ol> | ||
Line 32: | Line 49: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | Padlock0501 | ||
| align="center" | 10 uM | | align="center" | 10 uM | ||
| align="center" | 100 pmol total | | align="center" | 100 pmol total | ||
Line 60: | Line 77: | ||
<li>Heat kill enzyme with 15 minutes at 65C</li> | <li>Heat kill enzyme with 15 minutes at 65C</li> | ||
</ol> | </ol> | ||
****** | |||
<li>Template-Bead Binding</li> | <li>Template-Bead Binding</li> | ||
<ol type="A"> | <ol type="A"> | ||
Line 68: | Line 86: | ||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | ||
</ol> | </ol> | ||
****** | |||
<li>Padlock Hybridization</li> | <li>Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Make | <li>Make following 6.1X master mixes</li> | ||
<ol type=" | |||
<li> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
< | |- style="background-color:#DA9694;font-size:12pt;font-weight:bold" align="center" | ||
<li> | | width="150" height="39" | Reagent | ||
<li> | | width="85" | MM1 (uL) | ||
<li>Incubate at 37C for | | width="85" | MM2 (uL) | ||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Padlock0501 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 3.1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | PNK Reaction | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase Buffer | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
| align="center" align="center" valign="bottom" | 6.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 49.6 | |||
| align="center" align="center" valign="bottom" | 52.7 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 62 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 62 | |||
|} | |||
<li>Resuspend sample in 20 uL padlock buffer</li> | |||
<li>Incubate at 37C for 30 minutes</li> | |||
<li>Wash twice with wash buffer</li></ol> | |||
</ol> | |||
==Protocol-Part 2== | |||
<ol start="4"> | |||
<li>Circularization</li> | |||
<ol type="A"> | |||
<li>Prepare 6.5X 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="190" height="30" | Reagent | |||
| width="65" | Stock Conc | |||
| width="65" | Final Amount | |||
| width="65" | 1x Vol (uL) | |||
| width="105" | MM Vol (6.5x) (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" | 52 | |||
|- 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" | 3.9 | |||
|- 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" | 19.5 | |||
|- 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" | 13 | |||
|- 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" | 13 | |||
|- 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" | 20.8 | |||
|- 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" | 7.8 | |||
|- 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" | 130 | |||
|} | |||
<li>Add 20 uL phusion mix to 20 uL reaction</li> | |||
<li>Incubate at 55C for 2 hours</li> | |||
</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 1 hour at 37C</li> | |||
<li>Heat kill by incubating for 5 min at 95C</li> | |||
</ol></ol> | |||
Continued [[Daniel:Notebook/ComboLock/2017-2-28|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170227]] |
Latest revision as of 22:51, 27 February 2017
RCA + Circularization Test[edit]
Repeating the circularization and RCA test using the three different polymerase enzymes to classify which is best to use. This will use the C4-C2 template, which is the simplified template that is the equivalent of the two C probe arms ligated and gap-filled.
Protocol-Part 1[edit]
Sample Matrix
Sample | Condition |
Sample 1 (ABC) | Sample |
Sample 2 (ABC) | No Padlock PO4 |
- Phosphorylation-Padlock0501
- 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
- Dilute 5 uL C4-C2 template into 45 uL nfH2O
- Add 2 uL diluted template 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
- Padlock Hybridization
- Make following 6.1X master mixes
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 30 minutes
- Wash twice with wash buffer
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Polynucleotide Kinase Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
Padlock0501 | 10 uM | 100 pmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | MM1 (uL) | MM2 (uL) |
Padlock0501 | 0 | 3.1 |
PNK Reaction | 6.2 | 0 |
Amp Ligase Buffer | 6.2 | 6.2 |
nfH2O | 49.6 | 52.7 |
Total | 62 | 62 |
Protocol-Part 2[edit]
- Circularization
- Prepare 6.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 55C for 2 hours
- 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 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (6.5x) (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 |
Continued tomorrow