Daniel:Notebook/ComboLock/2016-8-18

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Revision as of 00:10, 18 August 2016 by >Djacobse (Created page with "=Positive Control Amplicon Test= <ol> <li>Latch and Padlock Hybridization</li> <ol type="A"> <li>Mix together samples according to table; use 100 uL LPH buffer with 100 nM Pa...")
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Positive Control Amplicon Test

  1. Latch and Padlock Hybridization
    1. Mix together samples according to table; use 100 uL LPH buffer with 100 nM Padlock0001-68
    2. Incubate for 1 hr at 37C
    3. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    4. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    5. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
      1. For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
    6. Incubate for 30 min at 37C
    7. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    8. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    9. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
  2. Circularization
    1. Prepare 20 uL KLN mix
    2. Prepare 4.1x master mix of Phusion mix
    3. Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
    4. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    5. Incubate at 55C overnight (XX hours)
    6. Continued tomorrow
    Sample No Sample Name LatchX2 LatchX3 PCAmp1 PCAmp3 Expected Product
    1 Amp1 Test X   X   Amp1
    2 Amp3 Test   X   X Amp3
    3 LatchX2 Specificity X   X X Amp1
    4 LatchX3 Specificity   X X X Amp3
    5 No Template X X     None
    6 No Latch     X X None
    7 No Padlock X X X X None