Daniel:Notebook/ComboLock/2017-3-25: Difference between revisions

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<ol start="3">
<ol start="3">
<li>TBE gel</li>
<li>TBE gel</li>
<ol type="A">
<li>Mix 64 uL TBE and 16 uL 6X dye</li>
<li>Mix 64 uL TBE and 16 uL 6X dye</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
Line 19: Line 20:
<li>Stain with 2 uL SYBR gold for 3 minutes</li>
<li>Stain with 2 uL SYBR gold for 3 minutes</li>
<li>Rinse and image in gel doc</li>
<li>Rinse and image in gel doc</li>
</ol></ol>
[[Image:2017-03-25-LinearProductProduction.png|400px]]
Still too much circular product present. I'll try one more time to make it linear.
==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">
<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>
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center"
| width="220" height="30" | Reagent
| width="105" | Single Rxn Vol
|- style="font-size:12pt"
| height="15"  valign="bottom" | Circular Product (1X)
| align="center" align="center" valign="bottom" | 4
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Bglii Cutter Primer (100 uM)
| align="center" align="center" valign="bottom" | 2
|- style="font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer 3.1
| align="center" align="center" valign="bottom" | 2
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Bglii
| align="center" align="center" valign="bottom" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 11
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Total
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20
|}
<li>Use the following thermocycler program</li>
<ol type="a">
<li>5 min 95C</li>
<li>Ramp to 50C at 0.2C/s</li>
<li>10 min 50C</li>
<li>4 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li>
<li>Heat kill with 65C for 20 min</li></ol>
</ol>
</ol>


==RCA Protocol==
Continued [[Daniel:Notebook/ComboLock/2017-3-26|tomorrow]]
 
 
 
[[Category:ComboLock]] [[Category:20170322]]

Latest revision as of 23:35, 30 March 2017

RCA Test[edit]

Back to Calendar

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]

  1. TBE gel
    1. Mix 64 uL TBE and 16 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
    4. File:GelLanes-20170325-LinearProduct.png
    5. Add 10 uL mix to gel lanes
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. 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.

  1. Bglii digest
    1. Make the following reactions; Do not add Bglii yet
    2. 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
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 4 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min

    Continued tomorrow