Daniel:Notebook/ComboLock/2017-4-29: Difference between revisions

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>Djacobse
>Djacobse
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|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | RCA Primer (1 uM each)
| height="15"  valign="bottom" | p12RC*** (10 uM)
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 0.2
| align="center" align="center" valign="bottom" | 20.4
| align="center" align="center" valign="bottom" | 2.04
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 1.24


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| height="15"  valign="bottom" | RCA Primer LLRC*** (10 uM)
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 0.2
| align="center" align="center" valign="bottom" | 51
| align="center" align="center" valign="bottom" | 2.04
| align="center" align="center" valign="bottom" | 31
| align="center" align="center" valign="bottom" | 1.24


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (10 mM)
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5.1
| align="center" align="center" valign="bottom" | 3.1
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer
| height="15"  valign="bottom" | 10X Buffer
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 54: Line 60:
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 12.4


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 10.2
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 6.2
| align="center" align="center" valign="bottom" | 0


|- style="font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | BSA (10 mg/mL)
| height="15"  valign="bottom" | BSA (10 mg/mL)
| align="center" align="center" valign="bottom" | 0.4
| align="center" align="center" valign="bottom" | 0.4
Line 66: Line 72:
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 5.6
| align="center" align="center" valign="bottom" | 11.7
| align="center" align="center" valign="bottom" | 57.12
| align="center" align="center" valign="bottom" | 119.34
| align="center" align="center" valign="bottom" | 37.2
| align="center" align="center" valign="bottom" | 75.02


|- style="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
|style="font-weight:bold" align="center" align="center" valign="bottom" | 163.2
|style="font-weight:bold" align="center" align="center" valign="bottom" | 153
|style="font-weight:bold" align="center" align="center" valign="bottom" | 99.2
|style="font-weight:bold" align="center" align="center" valign="bottom" | 93


|}
|}

Revision as of 20:21, 29 April 2017

Production Run (Started Yesterday)

Back to Calendar

Protocol-Part 4

  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 (10.2X) Master Mix Y (6.2X)
      Enzyme NA NEB Thermo
      Sample 4 0 0
      p12RC*** (10 uM) 0.2 2.04 1.24
      RCA Primer LLRC*** (10 uM) 0.2 2.04 1.24
      dNTP (10 mM) 0.5 5.1 3.1
      10X Buffer 2 20.4 12.4
      Phi29 1 0 0
      BSA (10 mg/mL) 0.4 4.08 0
      nfH2O 11.7 119.34 75.02
      Total 20 153 93
    3. Add 15 uL master mix to each tube
      1. Samples "A-C" get + master mixes; samples "D-F" get - master mixes
    4. Add 1 uM RCA primer and to new reaction tubes according to following rules
      1. "A,D" samples get 2 uL each LLRC & primer12RC
      2. "B,E" samples get 2 uL each LLRC*** & primer12RC
      3. "C,F" samples get 4 uL LLRC***
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 65 minutes at 20C to denature
  3. qPCR
    1. Make qPCR master mixes according to following recipe