Revision as of 21:09, 2 February 2017 by >Djacobse
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Phosphorothiorate RCA Primer (Started Yesterday)
- qPCR
- Make qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 17.2 uL AmpF6.4
- 430 uL Kapa SYBR Fast
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL AmpR indexed according to table
Sample
|
Sample
|
Condition
|
AmpR Index
|
Lane
|
Sample Vol (uL)
|
2X Kapa SYBR qPCR MM
|
10 uM Forward Primer
|
10 uM Reverse Primer
|
H2O
|
Total Volume (uL)
|
Sample 1AN
|
Sample
|
3 hours
|
20
|
A8
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 1BN
|
Sample
|
3 hours
|
20
|
A7
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 3AN
|
No C Probe
|
3 hours
|
21
|
A6
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 3BN
|
No C Probe
|
3 hours
|
21
|
A5
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 1AO
|
Sample
|
3 hours-Epi
|
22
|
A4
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 1BO
|
Sample
|
3 hours-Epi
|
22
|
A3
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 3AO
|
No C Probe
|
3 hours-Epi
|
23
|
A2
|
2
|
25
|
1
|
1
|
21
|
50
|
Sample 3BO
|
No C Probe
|
3 hours-Epi
|
23
|
A1
|
2
|
25
|
1
|
1
|
21
|
50
|
NTC
|
|
|
24
|
H1
|
0
|
25
|
1
|
1
|
23
|
50
|
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 94 uL TBE, 24 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Results
20170202-qPCR-RCATimeTest-Phosphorothiorate.png
2017-02-02-RCATimeTest-Phosphorothiorate.png
Version 2 Oligos Round 2
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Since I've been having so much trouble with the version 3 oligos, I want to test the v2 oligos again.
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-Phase 1
- Phosphorylation-MirrorC Probe B (PCCB-02)
- Set up reaction according to table
Reagent
|
Stock Conc
|
Final Conc./Amount
|
uL added
|
T4 Polynucleotide Kinase Buffer
|
10X
|
1X
|
2
|
ATP
|
10 mM
|
1 mM
|
2
|
PCCB-02
|
10 uM
|
100 pmol total
|
10
|
T4 DNA Kinase
|
10 U/uL
|
10 U
|
1
|
nfH2O
|
NA
|
NA
|
5
|
Total
|
|
|
20
|
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- 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
|
PCCA
|
1
|
8
|
Mirror C Probe B reaction
|
2
|
16
|
Wash buffer
|
2
|
16
|
Total
|
5
|
40
|
- 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; start part 4 after incubation begins
- 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
- Phosphorylation-Lock oligo
- Set up reaction according to table
Reagent
|
Stock Conc
|
Final Conc./Amount
|
uL added
|
T4 DNA Ligase Buffer
|
10X
|
1X
|
2
|
ATP
|
10 mM
|
1 mM
|
2
|
Lock0201
|
10 uM
|
10 pmol total
|
10
|
T4 DNA Kinase
|
10 U/uL
|
10 U
|
1
|
nfH2O
|
NA
|
NA
|
5
|
Total
|
|
|
20
|
- Incubate at 37C for 30 min
- Heat kill enzyme with 15 minutes at 65C
- Lock and Latch Hybridization/Ligation
- Set up the following master mix; Note: for sample 4 add un-phosphorylated Lock0201
Reagent
|
Stock Conc
|
Final Conc./Amt
|
uL added
|
Master Mix (6.2X)
|
Master Mix (2.2X)
|
T4 Ligase Reaction Buffer
|
5X
|
1X
|
4
|
24.8
|
8.8
|
Latch Oligo
|
10 uM
|
10 pmol
|
1
|
6.2
|
2.2
|
Lock Oligo Phosphate Reaction
|
(5 uM Lock)
|
10 pmol
|
2
|
12.4
|
*2.2*
|
nfH2O
|
NA
|
NA
|
12
|
86.8
|
39.2
|
Total
|
|
|
19
|
74.4
|
26.4
|
- Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
- Incubate at 95C for 5 min
- Lower temperature to 40C; 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 2 of the following master mix
- 8.2 uL 10 uM padlock0401 or Padlock0401A
- 8.2 uL Amp Ligase 10X reaction buffer
- 65.6 uL nfH2O
- Resuspend sample in 20 uL padlock buffer; incubate A samples with Padlock0401 and B samples with Padlock0401A
- 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 40C for 4 hours
Continued tomorrow
Buffers
Wash Buffer
Reagent
|
Stock
|
Final
|
Dilution
|
Amt in 20 mL
|
NaCl
|
1.5 M
|
0.5 M
|
3
|
6.66 mL
|
Tris-HCl
|
500 mM
|
20 mM
|
25
|
800 uL
|
EDTA
|
0.5M
|
1 mM
|
500
|
40 uL
|
nf H2O
|
NA
|
NA
|
NA
|
12.5 mL
|