Daniel:Notebook/ComboLock/2016-9-28

From ZhangLabWiki
Jump to navigation Jump to search

Positive Control Amplicon Production (PCAmp4)[edit]

Back to Calendar

Protocol[edit]

  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[edit]

    Sample ng/uL ssDNA
    C4-C2 Amplicon 557.1

    Protocol (Part 2)[edit]

    1. Size Select
      1. For sample lanes, mix 32 uL sample, 28 uL 6X loading dye and 100 uL TBE (160 uL total)
      2. For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
      3. Add 20 uL to each lane (2 gels total, 1 per sample)
      4. Run gel for 25 minutes at 250V
      5. While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube
      6. Stain gel for 3 minutes with 3 uL SYBR gold
      7. Image in gel doc
      8. Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)
      9. Image in gel doc post-extraction
      10. Centrifuge tubes at 12000rpm for 1.5 minutes
      11. Discard 0.5 mL tube and resuspend in 500 uL TE buffer
      12. Incubate at 37C overnight with vigorous shaking; continued tomorrow