Daniel:Notebook/ComboLock/2017-3-25: Difference between revisions
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>Djacobse |
>Djacobse |
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | Circular Product (1X) | ||
| align="center" align="center" valign="bottom" | 4 | | align="center" align="center" valign="bottom" | 4 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Bglii Cutter Primer ( | | height="15" valign="bottom" | Bglii Cutter Primer (100 uM) | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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|- style="font-size:12pt" | |- style="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" | 11 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
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</ol> | </ol> | ||
Continued [[Daniel:Notebook/ComboLock/2017-3-26|tomorrow]] | |||
[[Category:ComboLock]] [[Category: | [[Category:ComboLock]] [[Category:20170322]] |
Latest revision as of 23:35, 30 March 2017
RCA Test[edit]
This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.
Linear Product Production (Continued from yesterday)[edit]
- TBE gel
- Mix 64 uL TBE and 16 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot File:GelLanes-20170325-LinearProduct.png
- 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
File:2017-03-25-LinearProductProduction.png
Still too much circular product present. I'll try one more time to make it linear.
Linear Product Production[edit]
This time we'll start with the circular product and chop it.
- Bglii digest
- 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 1 uL Bglii
- Heat kill with 65C for 20 min
Reagent | Single Rxn Vol |
Circular Product (1X) | 4 |
Bglii Cutter Primer (100 uM) | 2 |
10X Buffer 3.1 | 2 |
Bglii | 1 |
nfH2O | 11 |
Total | 20 |
Continued tomorrow