Daniel:Notebook/ComboLock/2016-10-14
Jump to navigation
Jump to search
Padlock0301 Dilution Test (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 32.5 uL exonuclease I (20 units/μL) and 6.5 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Prepare the following master mix
- 334.25 uL nfH2O
- 9.55 uL 100 uM AmpF-CLv2
- 477.5 uL Kapa SYBR Fast MM
- Add 43 uL master mix to each well
- Add sample and AmpR indexes according to sample table in the next section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 176 uL TBE, 44 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
qPCR Sample Table[edit]
Sample | Template Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF-CLv2 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 6A | 10 amol | A1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6B | 10 amol | B1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6C | 10 amol | C1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8A | 100 zmol | D1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8B | 100 zmol | E1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8C | 100 zmol | F1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9A | 10 zmol | G1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9B | 10 zmol | H1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9C | 10 zmol | A12 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10A | 1 zmol | B12 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10B | 1 zmol | C12 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10C | 1 zmol | D12 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11A | 100 ymol | E12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11B | 100 ymol | F12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11C | 100 ymol | G12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12A | 10 ymol | H12 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12B | 10 ymol | A2 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12C | 10 ymol | B2 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | C2 | 26 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
- 20161014-qPCR-Padlock0301-Dilutions.png
qPCR Curve
- 2016-10-14-Padlock0301-Dilutions.png
Gel Image 1
- 2016-10-14-Padlock0301-Dilutions-Gel2.png
Gel Image 2
BSA Ab-Oligo Linkage[edit]
- Buffer Prep
- Mix 1.4 mL of monobasic and 3.6 mL of dibasic and 45 mL ddH2O to make 50 mL of 100 mM (buffer pH 7.2)
- Antibody Activation
- Resuspend antibodies at 1 mg/mL in PBS
- Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min
- Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate
- Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer
- Add 2 uL sulfo-SMCC to sample and 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
- Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA
- Add 1.3 uL oligonucleotide to tube (or plate)
- 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
I accidentally eluted the Ab fraction into a wash collector (with wash buffer in it), so I'm going to have to repeat the experiment on Monday
Cell Preparation[edit]
Prepping U87-MG cells for an experiment. Since I have a potential cold and I want to nurse it, I'll try and do the actual experiment on Monday.
- Fix cells
- Pellet cells at 600g for 3 min
- Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT
- Wash cells with wash buffer
- Pellet cells and permeabilize with 2 mL ice-cold methanol for 10 min on ice; split cells into individual 1.5 mL tubes
- Store cells in -80C