Revision as of 19:44, 16 March 2017 by >Djacobse
Circularization Comparison (T4 Poly/Ligase)[edit]
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Based on the March 8 experiment I will try the protocol again with a special note for testing against the bolt oligo and paying close attention to circularization.
Protocol[edit]
Sample Matrix
Sample
|
Condition
|
Sample 1 (AB)
|
Normal
|
Sample 2 (AB)
|
No Template
|
Sample 3 (AB)
|
No C probes
|
Sample 4 (AB)
|
Dephosphorylated
|
Sample 5 (AB)
|
Positive Control (C4-C2)
|
- Note:Because sample 5 is the finished product (ligated C probes), it will be started at step 5
******
- 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 0.2 mL tubes
Reagent
|
uL per Sample
|
Exp uL Total (1,2,4)
|
No C uL Total (3)
|
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; Start step 3 during this process
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
Protocol-Part 2[edit]
- Dephosphorylation (Bolt Oligo)
- Mix the following reaction
- 5 uL Bolt0301 (10 uM)
- 2 uL CutSmart Buffer (10X)
- 2 uL rSAP enzyme
- 16 uL nfH2O
- Incubate at 37C for 30 minutes
- Incubate at 65 for 5 minutes
******
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
Reagent
|
Stock Conc
|
Final Conc./Amt
|
uL added
|
Master Mix (6.2X)
|
Master Mix NP (2.2X)
|
T4 Ligase Reaction Buffer
|
5X
|
1X
|
4
|
24.8
|
8.8
|
Latch Oligo
|
10 uM
|
10 pmol
|
1
|
6.2
|
2.2
|
Bolt Oligo
|
10 uM
|
10 pmol
|
1
|
6.2
|
0
|
Dephosphorylated Bolt
|
2.5 uM
|
10 pmol
|
8
|
0
|
17.6
|
nfH2O
|
NA
|
NA
|
13
|
80.6
|
13.2
|
Total
|
|
|
19
|
117.8
|
41.8
|
- Add 19 uL probe mix to each sample
- Incubate at 55C for 10 minutes
- Incubate at 25C for 10 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
Protocol-Part 3[edit]
Sample Matrix 2-Circularization
Sample Code
|
Condition
|
Samples Included (#)
|
X
|
T4 Polymerase & Ligase
|
1-5 (10)
|
Y
|
Phusion + Amp Ligase
|
1,4 (4)
|
Z
|
Phusion + Amp Ligase-Hot Start
|
1,4 (4)
|
- Padlock Hybridization
- Wash samples one time and resuspend in 30 uL nfH2O
- Bake samples at 95 C and vortex to resuspend
- Note: Here I accidentally added the Phusion mix to the Y samples before adding the enzymes, so this is adjusted to only analyze the Z samples. Y samples will be processed tomorrow.
- Make the following master mixes
Reagent
|
MMX Unit
|
MMZ Unit
|
MMX (10.2X)
|
MMZ (4.2X)
|
Padlock0601 (10 uM)
|
1
|
1
|
10.2
|
4.2
|
Sample
|
5
|
5
|
0
|
0
|
Amp Ligase Buffer (10X)
|
0
|
2
|
0
|
8.4
|
5X T4 Ligase Buffer
|
4
|
0
|
40.8
|
0
|
dNTPs (1 mM)
|
2
|
0
|
20.4
|
0
|
nfH2O
|
7
|
12
|
71.4
|
50.4
|
Total
|
19
|
20
|
142.8
|
63
|
- Add 14 (X) or 15 (YZ) uL master mix to appropriate tubes
- Add 5 uL sample according to tables
- Incubate at 50C for 20 minutes
******
- Circularization
- For phusion samples, make the following master 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
|
- Follow the workflow in the below diagrams
File:CircProtocol-T4Poly.png
Continued tomorrow