Daniel:Notebook/ComboLock/2016-8-18
Jump to navigation
Jump to search
Positive Control Amplicon Test[edit]
Buffer Prep[edit]
Wash/Binding Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.5 M | 3 | 3.33 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 6.25 mL |
Elution Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Tris-HCl | 500 mM | 10 mM | 50 | 20 |
EDTA | 0.5M | 1 mM | 500 | 2 |
nf H2O | NA | NA | NA | 978 |
Low Salt Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
NaCl | 1.5 M | 0.15 M | 10 | 100 |
Tris-HCl | 500 mM | 20 mM | 25 | 40 |
EDTA | 0.5M | 1 mM | 500 | 2 |
nf H2O | NA | NA | NA | 858 |
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 2 |
dNTPs | 100 uM | 2 |
Hemo Klentaq | NA | 4 |
Amp Ligase | 5 U/uL | 2 |
nfH2O | NA | 10 |
Buffers[edit]
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 3 |
dNTPs | 100 uM | 3 |
Hemo Klentaq | NA | 6 |
Amp Ligase | 5 U/uL | 3 |
nfH2O | NA | 15 |
Protocol[edit]
- Latch and Padlock Hybridization
- Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer (total)
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution (see above table); incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68
- Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C overnight; continued tomorrow
Tube | Biotin-Amplicon | uL beads added | uL biotin probe added |
1 | Amp1 | 5 | 10 |
2 | Amp3 | 5 | 10 |
3 | PCAmp2 (C2) | 1 | 2 (dilute to 1 pmol/uL) |
Sample No | Sample Name | LatchX2 (300 ng - 2 uL 10 uM) | LatchX3 (300 ng - 2 uL 10 uM) | PCAmp1 | PCAmp3 | Expected Product |
1 | Amp1 Test | X | X | Amp1 | ||
2 | Amp3 Test | X | X | Amp3 | ||
3 | LatchX2 Specificity | X | X | X | Amp1 | |
4 | LatchX3 Specificity | X | X | X | Amp3 | |
5 | No Template | X | X | None | ||
6 | No Latch | X | X | None | ||
7 | No Padlock | X | X | X | X | None |
8 | PCAmp2 Only | X | X | None |