Daniel:Notebook/ComboLock/2017-6-5
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Sensitivity and Specificity Experiments[edit]
The purpose of this experiment will be to test the sensitivity and specificity of the ComboLock system using positive controls. The sample matrix is below, along with a column for rationales of why the sample exists in the first place. Note that
Sample No | Template | T1 C Probes | T2 C Probes | T1 Amt | T2 Amt | Test |
1 | T1 | X | 20 pmol | 0 | +++ Control | |
2 | T2 | X | 0 | 20 pmol | +++ Control-Does T2 work? | |
3 | AA | X | 0 | 0 | Negative Control | |
4 | BB | X | 0 | 0 | Negative Control | |
5 | T1 | X | 20 pmol | 0 | Negative Control-Specificity | |
6 | T2 | X | 0 | 20 pmol | Negative Control-Specificity | |
7 | T1/T2 50/50 | X | X | 10 pmol | 10 pmol | Orthogonality |
8 | T1/T2 10/90 | X | X | 2 pmol | 18 pmol | Sensitivity 10:1 |
9 | T1/T2 1/99 | X | 200 fmol | 19.8 pmol | Sensitivity 100:1 | |
10 | T1/T2 1/9999 | X | 2 fmol | 19.998 pmol | Sensitivity 10K:1 (1000 copies of transcript in 100K cells) |
Protocol[edit]
- Template dilutions
- Mix templates at appropriate ratios
- T1/T2 50/50: Mix 2 uL each oligo (10 uM each)
- T1/T2 10/90: Mix 4 uL each oligo (1 uM T1 and 10 uM T2)
- T1/T2 1/99: Mix 4 uL each oligo (100 nM T1 and 10 uM T2)
- T1/T2 1/9999: Mix 4 uL each oligo (1 nM T1 and 10 uM T2)
- To make dilutions, mix 5 uL sample in 45 uL nfH2O
- All samples are approximately 40 pmol added (2X bead loading)
- Pre-mix templates before bead binding
- Template-Bead Binding
- Suspend 2 uL (20 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Resuspend oligos in mixture of 20 uL Dynabuffer, 16 uL nfH2O
- 4 uL appropriate template oligo mix according to sample matrix(Total beads=20 ug=10 pmol available binding; 2X excess) 6X samples=12 uL
- Incubate at RT for 15 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 20 uL appropriate probe mixture to beads following sample matrix above
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
Reagent | uL per Sample | T1 Mix (6X) | T2 Mix (2X) | T1/T2 Mix (2X) |
PCCA-03 (10 uM) | 1 | 6 | 0 | 2 |
PCCB-03 (10 uM) | 1 | 6 | 0 | 2 |
PCCA-T2-03 (10 uM) | 1 | 0 | 2 | 2 |
PCCB-T2-03 (10 uM) | 1 | 0 | 2 | 2 |
DynaBeads Wash Buffer | 10 | 60 | 20 | 20 |
nfH2O | 8 | 48 | 16 | 12 |
Total | 20 | 120 | 40 | 40 |
Protocol-Part 2[edit]
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
- Pre-anneal latch/bolt
- Heat mix to 95C for 3 minutes
- Lower mix to 55C at rate of 0.2C/sec
- Incubate at 55C for 10 minutes
- Add 19 uL annealed 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
- Wash beads with 100 uL Dynabuffer
- Padlock Hybridization
- Add 20 uL of following 10.2X Master Mix:
- 10.2 uL 10 uM Padlock0601
- 20.4 uL 10X Amp Ligase buffer
- 173.4 uL nfH2O
- Incubate at 70C for 5 min
- Incubate at 50C for 15 minutes
- Wash once with wash buffer
- Resuspend in 20 uL 1X Amp Ligase buffer
- Circularization
- Make the following master mix
- Add 40 uL circularization mix to each sample
- Incubate at 55C for 4 hours
- Incubate at 95C for 5 minutes
- Hold at 16C
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (10.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 40.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 10.2 |
Bolt Oligo | 10 uM | 10 pmol | 1 | 10.2 |
nfH2O | NA | NA | 13 | 132.6 |
Total | 19 | 193.8 |
******
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (10.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 84 |
dNTP | 1 mM | 600 pmol | 0.6 | 6.3 |
Betaine | 5 M | 15 umol | 3 | 31.5 |
10X AmpLigase Buffer | 10X | 1X | 4 | 42 |
Amp Ligase | 5 U/uL | 10 U | 2 | 21 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 33.6 |
nf H2O | 19.2 | 201.6 | ||
Total | 40 | 420 |
Continued tomorrow