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

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[[Category:ComboLock]] [[Category:20160822]] [[Category:20160823]]
[[Category:ComboLock]] [[Category:20160928]]

Revision as of 00:57, 28 September 2016

Positive Control Amplicon Production (PCAmp4)

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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  

    Protocol (Part 2)

    1. TBE Gel
      1. Mix 32 uL TBE and 8 uL 6x Gel Loading dye
      2. Aliquot 10 uL for each sample to parafilm
      3. Add 2 uL of diluted sample/ladder to the drops
      4. Mix with the loading pipette and add 10 uL to each lane
      5. Run for 25 min at 250V
      6. Stain with 3 uL SYBR Gold for 3 min
      7. Rinse and image (see gallery below)

    [[Image:|500px]]

    1. Size Select
      1. For sample lanes, mix 16 uL sample, 14 uL 6X loading dye and 50 uL TBE (80 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 for 1 hour with vigorous shaker
      13. Centrifuge at 12000 rpm for 1.5 minutes
      14. Extract the supernatant and place into NanoSep column
      15. Centrifuge at 12000 rpm for 1.5 minutes
    2. Ethanol Precipitation
      1. Move sample to a fresh 2 mL tube
      2. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue
      3. Store at -80C overnight, continued tomorrow