Daniel:Notebook/ComboLock/2017-3-22: Difference between revisions

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>Djacobse
>Djacobse
Line 14: Line 14:
{| 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="background-color:#66FFFF;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#66FFFF;font-size:12pt;font-weight:bold" align="center"
| width="147" height="36" | Reagent
| width="220" height="36" | Reagent
| width="65" | Stock Conc
| width="100" | Stock Conc
| width="98" | Final Conc./Amount
| width="100" | Final Conc./Amount
| width="106" | uL added
| width="106" | uL added


Line 28: Line 28:
| height="15"  valign="bottom" | PosCon Circ Latch Oligo
| height="15"  valign="bottom" | PosCon Circ Latch Oligo
| align="center" | 100 uM
| align="center" | 100 uM
| align="center" | 500 umol
| align="center" | 200 pmol
| align="center" align="center" | 5
| align="center" align="center" | 1


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Padlock 0601 oligo
| height="15"  valign="bottom" | Padlock 0601 oligo
| align="center" | 100 uM
| align="center" | 100 uM
| align="center" | 500 umol
| align="center" | 200 pmol
| align="center" align="center" | 5
| align="center" align="center" | 1


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | C4 Positive Control oligo
| height="15"  valign="bottom" | C4 Positive Control oligo
| align="center" | 100 uM
| align="center" | 100 uM
| align="center" | 500 umol
| align="center" | 200 pmol
| align="center" align="center" | 5
| align="center" align="center" | 1


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | C2 positive Control oligo
| height="15"  valign="bottom" | C2 positive Control oligo
| align="center" | 100 uM
| align="center" | 100 uM
| align="center" | 500 umol
| align="center" | 200 pmol
| align="center" align="center" | 5
| align="center" align="center" | 1


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
Line 59: Line 59:
| align="center" | NA
| align="center" | NA
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 19
| align="center" align="center" | 35


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"

Revision as of 18:07, 22 March 2017

Circular Product Production

Back to Calendar

The goal of this will be to produce a large amount of circular product for use in testing.

Protocol

  1. Ligation
    1. Make reaction mixture according to table; Do not add ligase yet
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Ligase Buffer 5X 1X 10
      PosCon Circ Latch Oligo 100 uM 200 pmol 1
      Padlock 0601 oligo 100 uM 200 pmol 1
      C4 Positive Control oligo 100 uM 200 pmol 1
      C2 positive Control oligo 100 uM 200 pmol 1
      T4 DNA Ligase 5 U/uL 5 U 1
      nfH2O NA NA 35
      Total     50
    3. Use following thermocycler program
      1. Heat mixture to 95C for 5 min
      2. Lower temp by 0.2C per second to 55C
      3. Hold mixture at 55C for 15 minutes
      4. Lower temp to 20C by reducing 0.2C per second
      5. Hold at 20C
    4. Add 1 uL T4 DNA ligase
    5. Incubate at 20C for 30 minutes
  2. TBE gel
  3. Mix uL TBE and uL 6X dye
  4. Aliquot 10 uL on to parafilm per sample/ladder
  5. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
  6. Add 10 uL mix to gel lanes
  7. Run gel for 24 minutes at 230V
  8. Stain with 2 uL SYBR gold for 3 minutes
  9. Rinse and image in gel doc