Revision as of 21:15, 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. I'm also going to test ethanol precipitation post-exonuclease digestion. This is because exonucleases (and Amp ligase for that matter) do not properly degrade from simple heat inactivation (at least not in the short times we use). The added enzymes can potentially produce problems downstream, and the exonucleases in particular may be responsible for the RCA reactions showing weaker amplification than they should, since all the RCA product is single-stranded. So I'll use ethanol precipitation to extract only the DNA from the mix.
Sample Matrix and Workflow Diagram
Sample
|
Condition
|
Sample 1 (AB)
|
Normal
|
Sample 2 (AB)
|
No Template
|
Sample 3 (AB)
|
No C probes
|
Sample 4 (AB)
|
No phosphate activation
|
Workflow Diagram
File:20170207-CL-Workflow.png
Workflow diagram for the experiment. This experiment tests both the reduced temperatures as well as using ethanol precipitation post-exonuclease digestion.
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
- Add 70 uL H2O
- Transfer to a 1.5 mL tube
- Add 300 uL 100% EtOH, 30 uL 3M NaAOc, 1 uL glycoblue
- Incubate overnight at -80C
Continued tomorrow