Daniel:Notebook/ComboLock/2017-7-7: Difference between revisions

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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="110" height="30" | Reagent
|style="background-color:#8DB4E2" width="190" height="30" | Reagent
|style="background-color:#8DB4E2" width="95" | Single Rxn Vol
|style="background-color:#8DB4E2" width="95" | Single Rxn Vol
|style="background-color:#95B3D7" width="95" | Master Mix (10.2X)
|style="background-color:#95B3D7" width="95" | Master Mix (8.2X)


|- 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" | 7
| align="right" align="center" valign="bottom" | 7
| align="center" align="center" valign="bottom" | 0
| align="right" 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="right" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 20.4
| align="right" align="center" valign="bottom" | 16.4


|- 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="right" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 0
| align="right" 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" | 10
| align="right" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 102
| align="right" align="center" valign="bottom" | 82


|- 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="right" align="center" valign="bottom" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 122.4
|style="font-weight:bold" align="right" align="center" valign="bottom" | 98.4


|}
|}
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<li>Hold at 12C</ol>
<li>Hold at 12C</ol>
</ol>
</ol>
Continued [[Daniel:Notebook/ComboLock/2017-7-8|tomorrow]]


[[Category:ComboLock]] [[Category:20170706]]
[[Category:ComboLock]] [[Category:20170706]]

Latest revision as of 23:27, 7 July 2017

Oligo+Protein Test (Started Yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. Exonuclease Digestion
    1. Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 2 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent Single Rxn (uL) Master Mix X (8.2X)
      Enzyme NA Thermo
      Sample 4 0
      p12RC*** (10 uM) 0.2 1.64
      RCA Primer LLRC*** (10 uM) 0.2 1.64
      dNTP (10 mM) 0.5 4.1
      10X Buffer 2 16.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 3.28
      nfH2O 11.7 95.94
      Total 20 123
    3. Add 15 uL master mix to each tube
    4. Add 4 uL sample to appropriate tubes
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add 1 uL enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 65 minutes at 20C to denature
  3. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (8.2X)
      RCA Rxn 7 0
      10X Buffer 3.1 2 16.4
      Bglii 1 0
      nfH2O 10 82
      Total 20 98.4
    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
    8. Hold at 12C

Continued tomorrow