Revision as of 18:22, 17 January 2017 by >Djacobse
Version 3 Oligos[edit]
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The main difference between version 2 oligos and version 3 oligos is only adapter length. The version 3 oligos use the original 18bp adapters instead of the 15bp ones. This helps the annealing temperatures be high, which is better for enzymes such as Amp Ligase and Phusion. The other difference is that the version 3 oligos have phosphate groups on the 5'
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 (PCCB-Mirror-v2)
|
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 95C for 5 min
- 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)
- 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 4 hours
Continued tomorrow