Revision as of 00:10, 24 January 2017 by >Djacobse
Circularization Test
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Since the results from the last experiment seemed to indicate that RCA hasn't been working for me, I'm going to try RCA again using the old positive amplicon systems. For examples see Oct-5-2016 and Sept-30-2016. I'll be using PCAmp4
Sample Matrix
Tube No
|
Molecules Available
|
Amount
|
0 (AB)
|
1.00E+11
|
1 pmol
|
2 (AB)
|
1.00E+09
|
10 fmol
|
4 (AB)
|
1.00E+07
|
100 amol
|
6 (AB)
|
1.00E+05
|
1 amol
|
Protocol
- Phosphorylation
- In a 0.2 mL tube, add ingredients according to table
Reagent
|
Stock Conc
|
Final Conc./Amount
|
uL added
|
AmpLigase Reaction Buffer
|
10X
|
1X
|
2
|
ATP
|
10 mM
|
1 mM
|
2
|
PCAmp2
|
100 uM
|
1 nmol total
|
10
|
T4 DNA Kinase
|
10 U/uL
|
10 U
|
1
|
nfH2O
|
NA
|
NA
|
5
|
Total
|
|
|
20
|
- Incubate at 37C for 30 min
- Ligation
- Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
Reagent
|
Stock Conc
|
Final Conc./Amount
|
uL added
|
AmpLigase Reaction Buffer
|
10X
|
1X
|
4
|
Latch X4 Oligo
|
100 uM
|
500 umol
|
5
|
5' Amplicon Oligo
|
100 uM
|
500 umol
|
5
|
Amp Ligase
|
5 U/uL
|
5 U
|
1
|
Phosphate Reaction
|
NA
|
NA
|
10
|
nfH2O
|
NA
|
NA
|
25
|
Total
|
|
|
50
|
- Heat reaction to 95C for 5 min
- Lower temp to 55C
- Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
- Incubate at 55C for 2 hours
- Heat to 95 C to denature dsDNA
- Purify with ssDNA column
- ssDNA Column
- Add 100 uL Binding Buffer to the sample; mix well
- Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
- Add 150 uL 100% EtOH to flow through; mix well
- Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Centrifuge empty column at 14000 rpm for 2 minutes
- Transfer to empty 1.5mL centrifuge tube (low bind)
- Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute
Protocol (Part 2)
- Size Select
- For sample lanes, mix 32 uL sample, 28 uL 6X loading dye and 100 uL TBE (160 uL total)
- For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
- Add 20 uL to each lane (2 gels total, 1 per sample)
- Run gel for 25 minutes at 250V
- While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube
- Stain gel for 3 minutes with 3 uL SYBR gold
- Image in gel doc
- Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)
- Image in gel doc post-extraction
- Centrifuge tubes at 12000rpm for 1.5 minutes
- Discard 0.5 mL tube and resuspend in 500 uL TE buffer
- Incubate at 37C overnight with vigorous shaking; continued tomorrow
- Latch and Padlock Hybridization
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution; incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Add 2 uL per sample of 10 uM Padlock0501 to 20 uL 1X Amp ligase buffer
- Heat probes to 95C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with vigorous shaking
- 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