Daniel:Notebook/ComboLock/2017-3-25: Difference between revisions
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</ol></ol> | </ol></ol> | ||
[[Image:2017-03-25-LinearProductProduction.png|400px]] | |||
<ol> | Still too much circular product present. I'll try one more time to make it linear. | ||
<li> | |||
==Linear Product Production== | |||
This time we'll start with the circular product and chop it. | |||
<ol start="3"> | |||
<li>Bglii digest</li> | |||
<ol type="A"> | <ol type="A"> | ||
<li> | <li>Make the following reactions; '''Do not add Bglii yet'''</li> | ||
{| 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="background-color:# | |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="220" height="30" | Reagent | ||
| width=" | | width="105" | Single Rxn Vol | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Circular Product (1X) | | 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" | | | height="15" valign="bottom" | Bglii Cutter Primer (100 uM) | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
|- style="font-size:12pt" | |- style="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 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | Bglii | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
|- 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" | ||
| 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 | ||
|} | |} | ||
<li> | <li>Use the following thermocycler program</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>5 min 95C</li> | ||
<li> | <li>Ramp to 50C at 0.2C/s</li> | ||
<li>10 min 50C</li> | |||
<li> | <li>4 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> | ||
<li> | <li>Heat kill with 65C for 20 min</li></ol> | ||
<li> | </ol> | ||
Continued [[Daniel:Notebook/ComboLock/2017-3-26|tomorrow]] | |||
| | |||
[[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