Padlock0301 Test[edit]
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This is a test of the padlock0301, which replaces the AmpF/LinkerR region of the padlock with a custom primer set. This is because my experiments on 9-29, 9-30, 10-3, and 10-4, which test the v1 and v2 padlock systems using AmpF and AmpR individually. AmpF has proved particularly dirty so I have designed v3 of the padlocks without it.
Protocol[edit]
Stock Dilutions[edit]
Create the following dilutions of PCC Template by taking 10 uL stock and diluting with 90 uL nfH2O (note:zero is normal 10 uM stock)
Tube No
|
Dilution
|
Molecules Available
|
Amount
|
0
|
1
|
1.00E+12
|
10 pmol
|
1
|
10
|
1.00E+11
|
1 pmol
|
2
|
100
|
1.00E+10
|
100 fmol
|
3
|
1000
|
1.00E+09
|
10 fmol
|
4
|
10000
|
1.00E+08
|
1 fmol
|
5
|
1.00E+05
|
1.00E+07
|
100 amol
|
6
|
100000
|
1.00E+06
|
10 amol
|
Samples[edit]
Sample
|
C Probes-VIM (C1+C2)
|
C Probes-SOD1 (C1+C2)
|
Template
|
Dilution
|
Dilution Amount
|
Sample 1 (ABC)
|
X
|
X
|
X
|
0
|
10 pmol
|
Sample 2
|
X
|
X
|
|
NA
|
None
|
Sample 3
|
|
|
X
|
0
|
10 pmol
|
Sample 4 (ABC)
|
X
|
X
|
X
|
2
|
100 fmol
|
Sample 5 (ABC)
|
X
|
X
|
X
|
4
|
1 fmol
|
Sample 6 (ABC)
|
X
|
X
|
X
|
6
|
10 amol
|
Buffers[edit]
Reagent
|
Stock
|
uL added
|
Amp Ligase Buffer
|
10X
|
3
|
dNTPs
|
100 uM
|
3
|
Hemo Klentaq
|
NA
|
6
|
Amp Ligase
|
5 U/uL
|
3
|
nfH2O
|
NA
|
15
|
Experiment Protocol[edit]
- 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 template oligo 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 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- 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 Padlock0301 to each sample to a fresh 0.2 mL tube
- 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
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; 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 10 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow