Daniel:Notebook/ComboLock/2017-3-15
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Circularization Comparison (T4 Poly/Ligase)[edit]
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
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- 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
- 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
******
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 |
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
- 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
- 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
- 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
- Follow the workflow in the below diagrams File:CircProtocol-T4Poly.png Continued tomorrow
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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 |
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) |
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 |
******
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 |