Daniel:Notebook/ComboLock/2016-11-3: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 118: | Line 118: | ||
<li> continued [[Daniel:Notebook/ComboLock/2016-10-28|tomorrow]] </li> | <li> continued [[Daniel:Notebook/ComboLock/2016-10-28|tomorrow]] </li> | ||
</ol></ol> | </ol></ol> | ||
===Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="160" height="30" | Reagent | ||
| width="65" | Stock Conc | | width="65" | Stock Conc | ||
| width="65" | Final Amount | | width="65" | Final Amount |
Revision as of 20:03, 3 November 2016
Circularization Optimization Testing (Started Monday)
Library Prep (Started Yesterday)
- Ethanol Precipitation
- Centrifuge for 25 minutes at 12000 rpm at 4C
- Remove supernatant and add 750 uL 70% EtOH
- Centrifuge for 12 minutes at 12000 rpm at 4C
- Remove supernatant and spin in vacuum centrifuge for 10 minutes
- Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A-C by reusing the water
Results
Sample | ng/uL | ug in 30 uL |
Phusion-RCA Product | 58.8 | 1.8 |
Antibody Binding Test
Test for antibody binding and amplification. I'll use a similar protocol to 10-21 which uses takes time points but I'll also be using the standard beads protocol (some modifications).
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; take 10 uL as a time point
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; take 10 uL as a time point
- 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; take 10 uL as a time point
- Apply magnet and remove supernatant; Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; take 10 uL as a time point
- Apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 55C for 2 hours
- continued tomorrow
Results
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 | 2 | 21 |
Amp Ligase | 5 U/uL | 10 U | 2 | 21 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 33.6 |
nf H2O | 1.2 | 12.6 | ||
Total | 20 | 210 |