Daniel:Notebook/ComboLock/2017-6-15: Difference between revisions
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>Djacobse |
>Djacobse |
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| align="center" | 40 nmol | | align="center" | 40 nmol | ||
| align="center" align="center" | 8 | | align="center" align="center" | 8 | ||
| align="center" align="center" | | | align="center" align="center" | 52 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
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| align="center" | 600 pmol | | align="center" | 600 pmol | ||
| align="center" align="center" | 0.6 | | align="center" align="center" | 0.6 | ||
| align="center" align="center" | | | align="center" align="center" | 3.9 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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| align="center" | 15 umol | | align="center" | 15 umol | ||
| align="center" align="center" | 3 | | align="center" align="center" | 3 | ||
| align="center" align="center" | | | align="center" align="center" | 19.5 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
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| align="center" | 1X | | align="center" | 1X | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" | | | align="center" align="center" | 13 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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| align="center" | 10 U | | align="center" | 10 U | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
| align="center" align="center" | | | align="center" align="center" | 13 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height=" | | height="15" valign="bottom" | Phusion HF DNA Polymerase | ||
| align="center" | 2000 U/mL | | align="center" | 2000 U/mL | ||
| align="center" | 6.4U | | align="center" | 6.4U | ||
| align="center" align="center" | 3.2 | | align="center" align="center" | 3.2 | ||
| align="center" align="center" | | | align="center" align="center" | 20.8 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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| align="center" align="center" | | | align="center" align="center" | | ||
| align="center" align="center" | 1.2 | | align="center" align="center" | 1.2 | ||
| align="center" align="center" | | | align="center" align="center" | 7.8 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
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| align="center" align="center" | | | align="center" align="center" | | ||
|style="font-weight:bold" align="center" align="center" | 20 | |style="font-weight:bold" align="center" align="center" | 20 | ||
|style="font-weight:bold" align="center" align="center" | | |style="font-weight:bold" align="center" align="center" | 130 | ||
|} | |} |
Revision as of 15:47, 15 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 | 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 |