Daniel:Notebook/ComboLock/2017-3-15: Difference between revisions
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<li>C Probe Hybridization</li> | <li>C Probe Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Combine following into | <li>Combine following into 0.2 mL tubes</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| 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" | |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="190" height="30" | Reagent | ||
| width="85" | uL per Sample | | width="85" | uL per Sample | ||
| width=" | | width="90" | Exp uL Total (1,2,4) | ||
| width="85" | No C uL Total | | width="85" | No C uL Total (3) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | PCCA-03 (10 uM) | | height="15" valign="bottom" | PCCA-03 (10 uM) | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" | | | align="center" align="center" | 6 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | PCCB-03 (10 uM) | | height="15" valign="bottom" | PCCB-03 (10 uM) | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
| align="center" align="center" | | | align="center" align="center" | 6 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Wash buffer | | height="15" valign="bottom" | Wash buffer | ||
| align="center" align="center" | 3 | | align="center" align="center" | 3 | ||
| align="center" align="center" | | | align="center" align="center" | 18 | ||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
|- style="font-size:12pt;font-weight:bold" | |- style="background-color:#A6A6A6;font-size:12pt;font-weight:bold" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
| align="center" align="center" | 5 | | align="center" align="center" | 5 | ||
| align="center" align="center" | | | align="center" align="center" | 30 | ||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
Revision as of 18:50, 15 March 2017
Circularization Comparison (T4 Ligase)
Based on the March 8 experiment I will try the protocol again with a special note for testing against the bolt oligo and paying close attention to circularization.
Protocol
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | Dephosphorylated |
Sample 5 (AB) | Positive Control (C4-C2) |
- Note:Because sample 5 is the finished product (ligated C probes), it will be started at step 5
******
- 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 0.2 mL tubes
- 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; Start step 3 during this process
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
******
Reagent | uL per Sample | Exp uL Total (1,2,4) | No C uL Total (3) |
PCCA-03 (10 uM) | 1 | 6 | 0 |
PCCB-03 (10 uM) | 1 | 6 | 0 |
Wash buffer | 3 | 18 | 10 |
Total | 5 | 30 | 10 |
Protocol-Part 2
- Dephosphorylation (Bolt Oligo)
- Mix the following reaction
- 5 uL Bolt0301 (10 uM)
- 2 uL CutSmart Buffer (10X)
- 2 uL rSAP enzyme
- 16 uL nfH2O
- Incubate at 37C for 30 minutes
- Incubate at 65 for 5 minutes
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 10 minutes
- Incubate at 25C for 10 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 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Padlock Hybridization
- Make the following master mix
- 2 uL Padlock0601 (10 uM)
- 2 uL Amp Ligase 10X buffer
- 16 uL nfH2O
- Resuspend samples in 20 uL master mix
- Incubate at 50C for 30 minutes
******
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (6.2X) | Master Mix NP (2.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 24.8 | 8.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 6.2 | 2.2 |
Bolt Oligo | 10 uM | 10 pmol | 1 | 6.2 | 0 |
Dephosphorylated Bolt | 2.5 uM | 10 pmol | 8 | 0 | 17.6 |
nfH2O | NA | NA | 13 | 80.6 | 13.2 |
Total | 19 | 117.8 | 41.8 |
******
Protocol-Part 3
- Circularization