Daniel:Notebook/ComboLock/2017-2-27
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RCA + Circularization Test
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
- 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 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
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 |