Revision as of 00:25, 2 March 2017 by >Djacobse
Padlock0601 Test
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Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam)
Protocol-Part 1
Sample Matrix
Sample
|
Condition
|
Sample 1 (AB)
|
Normal
|
Sample 2 (AB)
|
No Template
|
Sample 3 (AB)
|
No C probes
|
Sample 4 (AB)
|
No phosphate on padlock (Padlock0501)
|
- 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 1-2,4)
|
uL Total (No C Probe)
|
PCCA-03 (10 uM)
|
1
|
6
|
0
|
PCCB-03 (10 uM)
|
1
|
6
|
0
|
Wash buffer
|
3
|
18
|
10
|
Total
|
5
|
30
|
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-Part 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 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes
- Wash twice with 100 uL wash buffer
- Padlock Hybridization
- Make the following master mixes
Reagent
|
1X
|
MMX
|
MM-NP
|
Padlock0501 (10 uM)
|
2
|
0
|
4.2
|
Padlock0601 (10 uM)
|
2
|
12.2
|
0
|
Amp Ligase Buffer (10X)
|
2
|
12.2
|
4.2
|
nfH2O
|
16
|
97.6
|
33.6
|
Total
|
20
|
122
|
42
|
- Resuspend samples 1-3 in 20 uL MMX and samples 4A,4B in 20 uL MMNP
- Incubate at 50C 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
Continued tomorrow