Version 2 Oligos[edit]
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The version 2 oligos have several changes. The first is that they are shorter. The C probes have a 20bp target matching region (from 24bp) and the adapters are 15bp (from 18bp). This makes the C probes exactly 60bp which means they are regular oligos (not Ultramers) and can be received one day after ordering. A second improvement is to include the idea of the lock oligo. Note, this is not a Lock nucleic acid, it's just a name. The lock oligo fits in between the two C probes, spanning the gap between them. In conjunction with the latch oligo, this theoretically means that we can ligate the C probes together without using polymerase. This will allow the padlock probe to act as a standard gap-fill padlock (instead of 2 small gaps) since it is one medium sized gap. I won't have time today to actually run this, so the following protocol will only be for reference to start when I get back.
Since I didn't have time before break this is just the suggested protocol to use once I get back.
Sample Matrix[edit]
Sample
|
Condition
|
Sample 1 (AB)
|
Normal
|
Sample 2 (AB)
|
No Template
|
Sample 3 (AB)
|
No C probes
|
Sample 4 (AB)
|
No phosphate activation
|
Protocol[edit]
- Phosphorylation-MirrorC Probe B (PCCB-02)
- Set up reaction according to table
Reagent
|
Stock Conc
|
Final Conc./Amount
|
uL added
|
T4 Polynucleotide Kinase Buffer
|
10X
|
1X
|
2
|
ATP
|
10 mM
|
1 mM
|
2
|
PCCB-02
|
10 uM
|
100 pmol total
|
10
|
T4 DNA Kinase
|
10 U/uL
|
10 U
|
1
|
nfH2O
|
NA
|
NA
|
5
|
Total
|
|
|
20
|
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- 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 10 uM 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; start part 3 after incubation begins
- 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
- Phosphorylation-Lock oligo
- Set up reaction according to table
Reagent
|
Stock Conc
|
Final Conc./Amount
|
uL added
|
T4 DNA Ligase Buffer
|
10X
|
1X
|
2
|
ATP
|
10 mM
|
1 mM
|
2
|
Lock0201
|
10 uM
|
10 pmol total
|
10
|
T4 DNA Kinase
|
10 U/uL
|
10 U
|
1
|
nfH2O
|
NA
|
NA
|
5
|
Total
|
|
|
20
|
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
Reagent
|
Stock Conc
|
Final Conc./Amt
|
uL added
|
Master Mix (8.2X)
|
T4 Ligase Reaction Buffer
|
10X
|
1X
|
2
|
16.4
|
Latch Oligo
|
10 uM
|
10 pmol
|
1
|
8.2
|
Lock Oligo Phosphate Reaction
|
(5 uM Lock)
|
10 pmol
|
2
|
16.4
|
nfH2O
|
NA
|
NA
|
14
|
114.8
|
Total
|
|
|
19
|
155.8
|
- Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
- Incubate at 95C for 5 min
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 30 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
- Padlock Hybridization
- Make following master mix
- 16.4 uL 10 uM padlock0401
- 16.4 uL Amp Ligase 10X reaction buffer
- 131.2 uL nfH2O
- Resuspend sample in 20 uL padlock buffer
- Circularization
- Prepare 9X uL Phusion mix
Buffers[edit]
Wash Buffer
Reagent
|
Stock
|
Final
|
Dilution
|
Amt in 20 mL
|
NaCl
|
1.5 M
|
0.5 M
|
3
|
6.66 mL
|
Tris-HCl
|
500 mM
|
20 mM
|
25
|
800 uL
|
EDTA
|
0.5M
|
1 mM
|
500
|
40 uL
|
nf H2O
|
NA
|
NA
|
NA
|
12.5 mL
|