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="191" height="30" | Reagent
|style="background-color:#8DB4E2" width="230" height="30" | Reagent
|style="background-color:#8DB4E2" width="65" | Single Rxn Vol
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol
|style="background-color:#95B3D7" width="65" | Master Mix (13.2X)
|style="background-color:#95B3D7" width="85" | Master Mix (13.2X)


|- 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" | 5
| 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" | Bglii Cutter Primer + (100 uM)
| 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" | 10X Buffer 3.1
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 26.4
|- style="background-color:#BFBFBF;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="background-color:#BFBFBF;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" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 184.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 158.4


|}
|}


<li>Aliquot 14 uL master mix into new tube</li>
<li>Aliquot 12 uL master mix into new tube</li>
<li>Add 5 uL appropriate sample</li>
<li>Add 7 uL appropriate sample</li>
<li>Use the following thermocycler program</li>
<li>Mix with vortexer, spin down, and add 1 uL Bglii</li>
<ol type="a">
<li>Incubate 2 hr 37C</li>
<li>2 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li>
<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]

Back to Calendar

Protocol[edit]

  1. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. 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
    3. Aliquot 12 uL master mix into new tube
    4. Add 7 uL appropriate sample
    5. Mix with vortexer, spin down, and add 1 uL Bglii
    6. Incubate 2 hr 37C
    7. Heat kill with 65C for 20 min
  2. TBE Gel
    1. Mix 120 uL 10X TBE and 40 uL 6X dye
    2. Aliquot 8 uL TBE/dye mix onto parafilm per sample
    3. Add 4 uL sample to sample lanes
    4. Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample
    5. Add 10 uL from aliquots into appropriate gel lanes
    6. Run gel for 25 minutes at 250V
    7. Stain with 2 uL SYBR gold for 3 min
    8. Rinse and image in gel doc