Revision as of 23:46, 27 January 2017 by >Djacobse
RCA Timing Test (v3 Oligos)[edit]
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I'm going to test the v3 oligos again, this time using longer RCA times than the other ones. This is from what I saw in the last experiment, which suggests to me that increased RCA time can potentially yield results. As such, I'll try several times to test the theory.
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 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[edit]
- 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 65C for 5 min; Note: changed from 95C due to this
- Lower temperature to 55C; incubate 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)
- Heat mixture to 95C for 5 minutes; Cooldown on ice
- 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[edit]
- 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
- Prepare master mix according to table below
Reagent
|
uL Added
|
Master Mix (16.2X)
|
Template
|
4
|
NA
|
RCA Primer (10 uM)
|
2.5
|
40.5
|
dNTP (1 mM)
|
2
|
32.4
|
10X Buffer
|
2
|
32.4
|
Phi29
|
1
|
NA
|
BSA (10 mg/mL)
|
0.4
|
6.48
|
nfH2O
|
8.1
|
131.22
|
Total
|
20
|
243
|
- Add 14 uL master mix to each tube
- Add sample and to new reaction tubes according to following table
Sample Code
|
Phi29 Source
|
Incubation Time (hrs)
|
Sample (1AB)
|
No Template (2AB)
|
No C Probes (3AB)
|
No PO4 (4AB)
|
X
|
NEB
|
3 hrs
|
X
|
X
|
|
|
Y
|
NEB
|
6 hrs
|
X
|
|
X
|
|
Z
|
NEB
|
12 hrs
|
X
|
|
|
X
|
Q
|
Epicentre
|
3 hrs
|
X
|
|
|
X
|
- Incubate at 30C for X hours (see table above)
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Continued tomorrow