Daniel:Notebook/ComboLock/2017-6-15: Difference between revisions
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="font-size:12pt;font-weight:bold" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
|style="background-color:#8DB4E2" width=" | |style="background-color:#8DB4E2" width="145" height="30" | Reagent | ||
|style="background-color:#8DB4E2" width="95" | Single Rxn Vol | |style="background-color:#8DB4E2" width="95" | Single Rxn Vol | ||
|style="background-color:#95B3D7" width="95" | Master Mix ( | |style="background-color:#95B3D7" width="95" | Master Mix (6.2X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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| height="15" valign="bottom" | 10X Buffer 3.1 | | height="15" valign="bottom" | 10X Buffer 3.1 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 12.4 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Bglii | | height="15" valign="bottom" | Bglii | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 6.2 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 62 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 80.6 | ||
|} | |} |
Revision as of 21:12, 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
- 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 2 hours at 37C
- Heat kill by incubating for 5 min at 95C
******
******
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 |
Protocol-Part 4
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Add 4 uL sample to appropriate tubes
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- Hold at 12C
Reagent | Single Rxn (uL) | Master Mix X (6.2X) |
Enzyme | NA | Thermo |
Sample | 4 | 0 |
p12RC*** (10 uM) | 0.2 | 1.24 |
RCA Primer LLRC*** (10 uM) | 0.2 | 1.24 |
dNTP (10 mM) | 0.5 | 3.1 |
10X Buffer | 2 | 12.4 |
Phi29 | 1 | 0 |
nfH2O | 11.7 | 72.54 |
Total | 20 | 90.52 |
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 12.4 |
Bglii | 1 | 6.2 |
nfH2O | 10 | 62 |
Total | 20 | 80.6 |