Daniel:Notebook/ComboLock/2016-9-28: Difference between revisions

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>Djacobse
>Djacobse
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| align="center" align="center" |  
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|style="font-weight:bold" align="center" align="center" | 50
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<li>Add amplicons according to following table</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
| width="65" height="45" | Sample
| width="65" | PCAmp1
| width="65" | PCAmp3
| width="65" | LatchX2
| width="65" | LatchX3
|- style="font-size:12pt"
| height="15" align="center" | AmpliconX1
| align="center" | X
| align="center"  valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center"  valign="bottom" | &nbsp;
|- style="background-color:#D9D9D9;font-size:12pt" align="center"
| height="15" | AmpliconX3
| align="center" | &nbsp;
| X
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| X


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Revision as of 00:50, 28 September 2016

Positive Control Amplicon Production (PCAmp4)

Back to Calendar

Protocol

  1. Phosphorylation
    1. In a 0.2 mL tube, add ingredients according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      AmpLigase Reaction Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      PCAmp2 100 uM 1 nmol total 10
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 5
      Total     20
    3. Incubate at 37C for 30 min
  2. Ligation
    1. Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
    2. Reagent Stock Conc Final Conc./Amount uL added
      AmpLigase Reaction Buffer 10X 1X 4
      Latch Oligo 100 uM 500 umol 5
      5' Amplicon Oligo 100 uM 500 umol 5
      Amp Ligase 5 U/uL 5 U 1
      Phosphate Reaction NA NA 10
      nfH2O NA NA 25
      Total     50
    3. Heat reaction to 95C for 5 min
    4. Lower temp to 55C
    5. Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
    6. Incubate at 55C for 2 hours
    7. Heat to 95 C to denature dsDNA
    8. Purify with ssDNA column
  3. ssDNA Column
    1. Add 100 uL Binding Buffer to the sample; mix well
    2. Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
    3. Add 150 uL 100% EtOH to flow through; mix well
    4. Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
    5. Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    6. Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    7. Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    8. Centrifuge empty column at 14000 rpm for 2 minutes
    9. Transfer to empty 1.5mL centrifuge tube (low bind)
    10. Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute

    Nanodrop Results

    Sample ng/uL ssDNA
    C4-C2 Amplicon