Daniel:Notebook/ComboLock/2017-4-12: Difference between revisions
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=RCA Double Deuce= | =RCA Double Deuce (Started [[Daniel:Notebook/ComboLock/2017-4-11|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
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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="230" 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 (13.2X) | |style="background-color:#95B3D7" width="85" | Master Mix (13.2X) | ||
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" | 7 | ||
| 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" | | | 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" | 26.4 | | align="center" align="center" valign="bottom" | 26.4 | ||
|- 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" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style="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" | 132 | | align="center" align="center" valign="bottom" | 132 | ||
|- style=" | |- 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" | 158.4 | ||
|} | |} | ||
<li>Aliquot | <li>Aliquot 12 uL master mix into new tube</li> | ||
<li>Add | <li>Add 7 uL appropriate sample</li> | ||
<li> | <li>Mix with vortexer, spin down, and add 1 uL Bglii</li> | ||
<li>Incubate 2 hr 37C</li> | |||
<li>2 hr 37C | |||
<li>Heat kill with 65C for 20 min</li></ol> | <li>Heat kill with 65C for 20 min</li></ol> | ||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 120 uL 10X TBE and 40 uL 6X dye</li> | |||
<li>Aliquot 8 uL TBE/dye mix onto parafilm per sample</li> | |||
<li>Add 4 uL sample to sample lanes</li> | |||
<li>Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample</li> | |||
<li>Add 10 uL from aliquots into appropriate gel lanes</li> | |||
<li>Run gel for 25 minutes at 250V</li> | |||
<li>Stain with 2 uL SYBR gold for 3 min</li> | |||
<li>Rinse and image in gel doc</li></ol> | |||
</ol> | </ol> | ||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2017-04-12-RCADD-NEB.png|Samples amplified with NEB Phi29 | |||
File:2017-04-12-RCADD-Thermo.png|Samples amplified with Thermo Phi29 | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20170411]] |
Latest revision as of 20:55, 12 April 2017
RCA Double Deuce (Started Yesterday)[edit]
Protocol[edit]
- 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
- TBE Gel
- Mix 120 uL 10X TBE and 40 uL 6X dye
- Aliquot 8 uL TBE/dye mix onto parafilm per sample
- Add 4 uL sample to sample lanes
- Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample
- Add 10 uL from aliquots into appropriate gel lanes
- Run gel for 25 minutes at 250V
- Stain with 2 uL SYBR gold for 3 min
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (13.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 26.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 132 |
Total | 20 | 158.4 |
- 2017-04-12-RCADD-NEB.png
Samples amplified with NEB Phi29
- 2017-04-12-RCADD-Thermo.png
Samples amplified with Thermo Phi29