Daniel:Notebook/ComboLock/2017-4-4: Difference between revisions

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Line 12: Line 12:
{| 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="210" height="42" | Reagent
|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" | 8
| 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" | 2
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 12.4
| 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" | 1
| 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" | 9
| align="center" align="center" valign="bottom" | 24
| align="center" align="center" valign="bottom" | 55.8
| 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" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 40
|style="font-weight:bold" align="center" align="center" valign="bottom" | 68.2
|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>1 hr 37C; '''When this cycle starts add 2 uL Bglii'''</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]

Back to Calendar

Bgl Digestion-Excessive[edit]

  1. Bgl Digestion
    1. Make the following reactions; Do not add Bglii yet
    2. 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
    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 2 uL Bglii
      5. Heat kill with 65C for 20 min
  2. TBE Gel
    1. Mix 60 uL 10X TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
    3. Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. Run gel for 24 minutes at 230V
    6. Stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc

File:2017-04-05 -RCATest-CPLP-ssSynthesis-sssThenBgl4hr.png