Revision as of 19:25, 8 November 2016 by >Djacobse
Circularization Optimization Testing (Started Monday)[edit]
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- 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[edit]
Sample
|
ng/uL
|
ug in 30 uL
|
Phusion-RCA Product
|
58.8
|
1.8
|
Antibody Binding Test[edit]
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
- Heat kill by incubating at 94C for 5 minutes
- Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
- Prepare 8.1X Master mix according to table below
- Add sample to new reaction tubes according to following table
Reagent
|
uL Added
|
Master Mix (10.2X)
|
Template
|
5
|
NA
|
RCA Primer (100 uM)
|
2.5
|
25.5
|
dNTP (1 mM)
|
0.8
|
8.16
|
10X Buffer
|
2
|
20.4
|
Phi29
|
1
|
10.2
|
nfH2O
|
8.7
|
88.74
|
Total
|
20
|
153
|
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes
- Hold at 10C; continued tomorrow
Results[edit]
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
|