Daniel:Notebook/ComboLock/2016-7-5
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Sequential Hybridization Test[edit]
Today I'm going to try testing the latch and padlock hybridizations sequentially. This is because of the unusual results of the C probe tests from and June 30 and July 2.
Protocol[edit]
Buffer Prep[edit]
C Probe Hyridization (CPH) Buffer
Reagent | Stock Conc. | Final Conc. | Amt. Added | Amt Added |
SSC | 20X | 1X | 50 uL | 500 uL |
Tween 20 | 100% | 0.10% | 1 uL | 10 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1 uL | 10 uL |
Riboside Vanadyl Complex | 200 mM | 20 mM | 100 uL | 1 mL |
Poly Vinylsulfonic Acid | 25% | 2.50% | 100 uL | 1 mL |
Salmon Sperm | 10 mg/mL | 100 ug/mL | 10 uL | 100 uL |
Probes | 100 uM | 100 nM | 1 uL/probe | *** |
Water | NA | NA | 740 uL | 7.4 mL |
Total | 1 mL | 10 mL |
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
PBS | 10X | 1X | 10 | 100 uL |
SSC | 20X | 4X | 5 | 200 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
nf H2O | NA | NA | NA | 700 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 1 uL |
SSC | 20X | 1X | 20 | 50 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
PBS | 10X | 1X | 10 | 100 uL |
nf H2O | NA | NA | NA | 850 uL |
Experiment[edit]
- C-Probe Hybridization
- Transfer cells to a new tube such that there are 100000 cells present
- Human-1000 uL; Mouse-85 uL
- Human cells from July 2; 1.17E5 cells/mL
- Pellet cells via centrifugation at 600g for 3 min
- Combine 1.5 uL of each C probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
- Prep
- Resuspend SOD1-PA in 63 uL nfH2O (1 mM)
- Resuspend SOD1-PB in 47 uL nfH2O (1 mM)
- Antibody Activation
- Resuspend SOD1 with 100 uL PBS (final conc 1 mg/mL)
- Do this twice:
- Equilibrate a 40 kDa spin column 4 times with 100 uL PBS, (100 mM pH 7.3)
- Spin at 1000xg for 2 min
- Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube
- Add 2 uL 3.3 mM sulfo-SMCC in 100 mM phosphate buffer to sample
- Stock is 100 mM sulfo-SMCC in 100 mM phosphate buffer
- Mix 3.3 uL stock with 96 uL 100 mM phosphate buffer (dilute from 1 M stock)
- Incubate for 2 hr at 4C with 3 times intermittent mixing
- Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
- Transfer antibodies to new column and spin for 3 min at 1000xg
- Oligonucleotide Activation
- For VIM-PA and VIM-PB:
- Add 1.3 uL 100 uM oligonucleotide to tube
- Add 2.2 uL 40 mM DTT
- Incubate at 95C for 2 min followed by 1 hr at 37C
- Add 20 uL PBS with 20 mM EDTA
- Remove excess DTT using two consecutive Zeba 7 kDa columns equilibrated with 100 mM PBS (10X PBS)
- Spin at 1500xg for 1 minute for equilibrium; 2 min for sample
- Ab-Oligo Mixing
- For VIM-PA and VIM-PB:
- Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)
- Amounts to 10X molar excess of oligo to Ab
- Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
- Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
- Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl
C Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | U87MG | 3T3 Cells | |
Sample 1 | X | X | X | |
Sample 2 | X | |||
Sample 3 | X | X | X | |
Sample 4 | X | |||
Sample 5 | X | X |
SOD1 Antibody Prep[edit]
Since I have the VIM antibody and C Probes ready and only the SOD1 C Probe, it makes sense to have this antibody as well.