Daniel:Notebook/ComboLock/2017-3-1: Difference between revisions
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>Djacobse (Created page with "=Padlock0601 Test= Back to Calendar Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam) ==Prot...") |
>Djacobse |
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==Protocol-Part 2== | ==Protocol-Part 2== | ||
<ol start="3"> | |||
<li>Lock and Latch Hybridization/Ligation</li> | |||
<ol type="A"> | |||
<li>Set up the following master mix</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | |||
| width="130" height="30" | Reagent | |||
| width="85" | Stock Conc | |||
| width="85" | Final Conc./Amt | |||
| width="85" | uL added | |||
| width="85" | Master Mix (8.2X) | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | T4 Ligase Reaction Buffer | |||
| align="center" | 5X | |||
| align="center" | 1X | |||
| align="center" align="center" | 4 | |||
| align="center" align="center" valign="bottom" | 32.8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Latch Oligo | |||
| align="center" | 10 uM | |||
| align="center" | 10 pmol | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" valign="bottom" | 8.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Lock Oligo | |||
| align="center" | 10 uM | |||
| align="center" | 10 pmol | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" valign="bottom" | 8.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 13 | |||
| align="center" align="center" valign="bottom" | 106.6 | |||
|- style="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" | 19 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 155.8 | |||
|} | |||
<li>Add 19 uL probe mix to each sample</li> | |||
<li>Incubate at 55C for 20 minutes</li> | |||
<li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> | |||
<li>Add 1 uL T4 Ligase to each reaction</li> | |||
<li>Incubate at RT for 30 min</li> | |||
<li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | |||
<li>Wash twice with 100 uL wash buffer</li></ol> | |||
****** | |||
<li>Padlock Hybridization</li> | |||
<ol type="A"> | |||
<li>Make the following 8.1X master mix (1X)</li> | |||
<ol type="a"> | |||
<li>16.2 uL 10 uM padlock0501(2uL)</li> | |||
<li>16.2 uL Amp Ligase 10X reaction buffer(2uL)</li> | |||
<li>129.6 uL nfH2O</li>(16uL)</ol> | |||
<li>Resuspend sample in 20 uL padlock buffer</li> | |||
<li>Incubate at 37C for 30 minutes</li></ol> | |||
****** | |||
<li>Circularization</li> | |||
<ol type="A"> | |||
<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="150" height="30" | Reagent | |||
| width="85" | Stock Conc | |||
| width="85" | Final Amount | |||
| width="85" | 1x Vol (uL) | |||
| width="85" | 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="15" 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 | |||
|} | |||
<li>Add 20 uL phusion mix to 20 uL reaction</li> | |||
<li>Incubate at 55C for 2 hours</li> | |||
</ol></ol> | |||
****** |
Revision as of 19:28, 1 March 2017
Padlock0601 Test
Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam)
Protocol-Part 1
- 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-Part 2
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 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)
- 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 |