Daniel:Notebook/ComboLock/2016-11-15
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BSA Beads Test
Given the results from the primers test and most recent cells test, it seems that using the primer from Chris with RCA has provided a more consistent positive control. I'm going to repeat the experiment from November 3rd regarding the BSA-strep beads.
Protocol
Sample | BSA Ab | BSA-Biotin | Dilution | Dilution Amount |
Sample 1 (AB) | X | X | 0 | 2 ug |
Sample 2 (AB) | X | NA | None | |
Sample 3 (AB) | X | 0 | 2 ug | |
Sample 4 (AB) | X | X | 2 | 20 ng |
Sample 5 (AB) | X | X | 4 | 200 pg |
- 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 2 mg/mL biotin-BSA 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 2 uL of BSA Ab-AB mix (75 ug/uL stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Incubate at RT for 1.5 hours with agitation
- Add 100 uL of wash buffer
- 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
- Latch and Padlock Hybridization
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube
- Tech Rep A-Latch0001
- Tech Rep B-Latch0002
- 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 with intermittent mixing
- Add 100 uL wash buffer
- 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 Phusion mix Phusion Mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 55C for 2 hours
- Heat kill by incubating at 94C for 5 minutes; tomorrow
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (10.2x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 81.6 |
dNTP | 1 mM | 600 pmol | 0.6 | 6.12 |
Betaine | 5 M | 15 umol | 3 | 30.6 |
10X AmpLigase Buffer | 10X | 1X | 2 | 20.4 |
Amp Ligase | 5 U/uL | 10 U | 2 | 20.4 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 32.64 |
nf H2O | 1.2 | 12.24 | ||
Total | 20 | 204 |
Library Prep
- Size Select Gel
- Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample (Samples 1AA, 1BA, 2AB, 2BB)
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 24 uL per sample/ladder lane into each well
- Run gel for 23 minutes at 225V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2016-11-15-CellRCA-SizeSelect.png
Before Image
- 2016-11-15-CellRCA-SizeSelectAfter.png
After Image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate 4 hours at 37C with vigorous shaking;
- Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
- Extract supernatant and add to nanosep column
- Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
- Incubate at -80C overnight; continued tomorrow