Daniel:Notebook/ComboLock/2017-3-1
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Padlock0601 Test[edit]
Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam)
Protocol-Part 1[edit]
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | No phosphate on padlock (Padlock0501) |
- 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 1-2,4) | uL Total (No C Probe) |
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[edit]
- 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 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
- Padlock Hybridization
- Make the following master mixes
- Resuspend samples 1-3 in 20 uL MMX and samples 4A,4B in 20 uL MMNP
- Incubate at 50C 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 | 1X | MMX | MM-NP |
Padlock0501 (10 uM) | 2 | 0 | 4.2 |
Padlock0601 (10 uM) | 2 | 12.2 | 0 |
Amp Ligase Buffer (10X) | 2 | 12.2 | 4.2 |
nfH2O | 16 | 97.6 | 33.6 |
Total | 20 | 122 | 42 |
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 |
Continued tomorrow