Daniel:Notebook/ComboLock/2017-2-27
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RCA + Circularization Test[edit]
Repeating the circularization and RCA test using the three different polymerase enzymes to classify which is best to use. This will use the C4-C2 template, which is the simplified template that is the equivalent of the two C probe arms ligated and gap-filled.
Protocol-Part 1[edit]
Sample Matrix
Sample | Condition |
Sample 1 (ABC) | Sample |
Sample 2 (ABC) | No Padlock PO4 |
- Phosphorylation-Padlock0501
- Set up reaction according to table
- 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
- Dilute 5 uL C4-C2 template into 45 uL nfH2O
- Add 2 uL diluted template 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
- Padlock Hybridization
- Make following 6.1X master mixes
- Resuspend sample in 20 uL padlock buffer
- Incubate at 37C for 30 minutes
- Wash twice with wash buffer
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Polynucleotide Kinase Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
Padlock0501 | 10 uM | 100 pmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | MM1 (uL) | MM2 (uL) |
Padlock0501 | 0 | 3.1 |
PNK Reaction | 6.2 | 0 |
Amp Ligase Buffer | 6.2 | 6.2 |
nfH2O | 49.6 | 52.7 |
Total | 62 | 62 |
Protocol-Part 2[edit]
- Circularization
- Prepare 6.5X uL Phusion mix
- Add 20 uL phusion mix to 20 uL reaction
- Incubate at 55C for 2 hours
- Exonuclease Digestion
- Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (6.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 52 |
dNTP | 1 mM | 600 pmol | 0.6 | 3.9 |
Betaine | 5 M | 15 umol | 3 | 19.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 13 |
Amp Ligase | 5 U/uL | 10 U | 2 | 13 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 20.8 |
nf H2O | 1.2 | 7.8 | ||
Total | 20 | 130 |
Continued tomorrow