Daniel:Notebook/ComboLock/2017-4-4: Difference between revisions
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>Djacobse |
>Djacobse |
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(3 intermediate revisions by the same user not shown) | |||
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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="190" height="30" | Reagent | ||
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol | |style="background-color:#8DB4E2" width="85" | Single Rxn Vol | ||
|style="background-color:#95B3D7" width="85" | Master Mix (6.2X) | |style="background-color:#95B3D7" width="85" | Master Mix (6.2X) | ||
Line 18: | Line 18: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | RCA Rxn | | height="15" valign="bottom" | RCA Rxn | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 10 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 10X Buffer 3.1 | | height="15" valign="bottom" | 10X Buffer 3.1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 4 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 24.8 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Bglii | | height="15" valign="bottom" | Bglii | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- 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" | | | align="center" align="center" valign="bottom" | 24 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 148.8 | ||
|- 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" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 40 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 173.6 | ||
|} | |} | ||
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<li>Ramp to 50C at 0.2C/s</li> | <li>Ramp to 50C at 0.2C/s</li> | ||
<li>10 min 50C</li> | <li>10 min 50C</li> | ||
<li> | <li>4 hr 37C; '''When this cycle starts add 2 uL Bglii'''</li> | ||
<li>Heat kill with 65C for 20 min</li></ol> | <li>Heat kill with 65C for 20 min</li></ol> | ||
</ol> | </ol> | ||
Line 61: | Line 61: | ||
<li>Rinse and image in gel doc</li> | <li>Rinse and image in gel doc</li> | ||
</ol></ol> | </ol></ol> | ||
[[Image:2017-04-05_-RCATest-CPLP-ssSynthesis-sssThenBgl4hr.png|500px]] | |||
[[Category:ComboLock]] [[Category:20170324]] |
Latest revision as of 19:17, 5 April 2017
RCA Test (Started March 24)[edit]
Bgl Digestion-Excessive[edit]
- Bgl Digestion
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 4 hr 37C; When this cycle starts add 2 uL Bglii
- Heat kill with 65C for 20 min
- TBE Gel
- Mix 60 uL 10X TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
- Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 10 | 0 |
10X Buffer 3.1 | 4 | 24.8 |
Bglii | 2 | 0 |
nfH2O | 24 | 148.8 |
Total | 40 | 173.6 |