Daniel:Notebook/ComboLock/2017-6-15: Difference between revisions
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>Djacobse (Created page with "=Antibody Detection Test-Positive Control (Started Yesterday)= Back to Calendar= ==Protocol-Part 3== <...") |
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==Protocol-Part 3== | ==Protocol-Part 3== | ||
<ol start=" | <ol start="3"> | ||
<li>Latch and Bolt Hybridization/Ligation</li> | |||
<ol type="A" start="8"> | |||
<li>Heat kill enzyme by incubating at 65C for 10 minutes</li> | |||
</ol> | |||
****** | |||
<li>Padlock Hybridization</li> | <li>Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> |
Revision as of 22:03, 14 June 2017
Antibody Detection Test-Positive Control (Started Yesterday)
Protocol-Part 3
- Latch and Bolt Hybridization/Ligation
- Heat kill enzyme by incubating at 65C for 10 minutes
- Padlock Hybridization
- Add 20 uL of following 6.2X Master Mix
- 6.2 uL 10 uM Padlock0601
- 12.4 uL 10X Amp Ligase buffer
- 105.4 uL nfH2O
- Incubate at 70C for 5 min
- Incubate at 50C for 15 minutes
- Wash once with wash buffer*Do not wash sample 5
- Resuspend in 20 uL 1X Amp Ligase buffer
- Circularization
- For phusion samples, make the following master mix
- Add 20 uL circularization mix to each sample
- Incubate at 55C for 2 hours
- Incubate at 95C for 5 minutes
- Hold at 16C
******
******
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (10.5x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 84 |
dNTP | 1 mM | 600 pmol | 0.6 | 6.3 |
Betaine | 5 M | 15 umol | 3 | 31.5 |
10X AmpLigase Buffer | 10X | 1X | 2 | 21 |
Amp Ligase | 5 U/uL | 10 U | 2 | 21 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 33.6 |
nf H2O | 1.2 | 12.6 | ||
Total | 20 | 210 |