Revision as of 00:06, 8 February 2017 by >Djacobse
v2 Oligos-Round 3
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Based on yesterday's conclusions, I've redesigned the experimental protocol to be more sensitive about temperature. The biotin-streptavidin bond is best kept under 70C. I'll use the v3 oligos for now, and try again with v2 if we have a problem.
Sample Matrix
Sample
|
Condition
|
Sample 1 (AB)
|
Normal
|
Sample 2 (AB)
|
No Template
|
Sample 3 (AB)
|
No C probes
|
Sample 4 (AB)
|
No phosphate activation
|
Protocol-Part 1
- 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 following into a 0.2 mL tube
Reagent
|
uL per Sample
|
uL Total (Samples+NT)
|
uL Total (No Phosphate)
|
uL Total (No C Probe)
|
PCCA-03 (10 uM)
|
1
|
4
|
2
|
0
|
PCCB-03 (10 uM)
|
1
|
4
|
2***
|
0
|
Wash buffer
|
3
|
12
|
6
|
10
|
Total
|
5
|
20
|
10
|
10
|
- 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
Protocol-Phase 2
- 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
|
5X
|
1X
|
4
|
32.8
|
Latch Oligo
|
10 uM
|
10 pmol
|
1
|
8.2
|
Lock Oligo
|
10 uM
|
10 pmol
|
1
|
8.2
|
nfH2O
|
NA
|
NA
|
13
|
106.6
|
Total
|
|
|
19
|
155.8
|
- Add 19 uL probe mix to each sample
- Incubate at 55C for 20 minutes
- 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 the following 8.1X master mix (1X)
- 16.2 uL 10 uM padlock0501(2uL)
- 16.2 uL Amp Ligase 10X reaction buffer(2uL)
- 129.6 uL nfH2O
(16uL)
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 30 minutes
- Circularization
- Prepare 8.5X uL Phusion mix
Reagent
|
Stock Conc
|
Final Amount
|
1x Vol (uL)
|
MM Vol (8.5x) (uL)
|
NAD+
|
5 mM
|
40 nmol
|
8
|
68
|
dNTP
|
1 mM
|
600 pmol
|
0.6
|
5.1
|
Betaine
|
5 M
|
15 umol
|
3
|
25.5
|
10X AmpLigase Buffer
|
10X
|
1X
|
2
|
17
|
Amp Ligase
|
5 U/uL
|
10 U
|
2
|
17
|
Phusion HF DNA Polymerase
|
2000 U/mL
|
6.4U
|
3.2
|
27.2
|
nf H2O
|
|
|
1.2
|
8.4
|
Total
|
|
|
20
|
140
|
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 55C for 2 hours
Protocol-Part3
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Take 10 uL and set aside for future testing
- Prepare 8.2X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
Reagent
|
uL Added
|
Master Mix (8.2X)
|
Template
|
3
|
0
|
RCA Primer (10 uM)
|
2.5
|
20.5
|
dNTP (1 mM)
|
0.8
|
6.56
|
10X Buffer
|
2
|
16.4
|
Phi29
|
1
|
0
|
nfH2O
|
10.7
|
87.74
|
Total
|
20
|
131.2
|
- Incubate at 30C for 6 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- Ethanol Precipitation
Continued tomorrow