Daniel:Notebook/ComboLock/2016-10-28

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Revision as of 17:15, 28 October 2016 by >Djacobse (Created page with "=Beads UMI Test= Back to Calendar ==Protocol== <ol start="2"> <li>Circularization</li> <ol type="A"> <li>Heat inactivate enzyme by incubating ...")
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Beads UMI Test

Back to Calendar

Protocol

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table in the next section
    2. Make master mixes according to following recipes
      1. 338.1 uL nfH2O
      2. 402.5 uL SYBR Fast
      3. 16.1 uL 10 uM AmpF
    3. Add 43 uL master mix to each well
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  4. TBE Gel
    1. Mix 160 uL TBE, 40 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

qPCR Sample Table

Sample Latch Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
Sample 1A Latch0001 A1 20 2 25 1 1 21 50
Sample 1B Latch0002 B1 20 2 25 1 1 21 50
Sample 1C LatchX4 C1 20 2 25 1 1 21 50
Sample 2A Latch0001 D1 21 2 25 1 1 21 50
Sample 2B Latch0002 E1 21 2 25 1 1 21 50
Sample 2C LatchX4 F1 21 2 25 1 1 21 50
Sample 3A Latch0001 G1 22 2 25 1 1 21 50
Sample 3B Latch0002 H1 22 2 25 1 1 21 50
Sample 3C LatchX4 A12 22 2 25 1 1 21 50
Sample 4A Latch0001 B12 23 2 25 1 1 21 50
Sample 4B Latch0002 C12 23 2 25 1 1 21 50
Sample 4C LatchX4 D12 23 2 25 1 1 21 50
Sample 5A Latch0001 E12 24 2 25 1 1 21 50
Sample 5B Latch0002 F12 24 2 25 1 1 21 50
Sample 5C LatchX4 G12 24 2 25 1 1 21 50
NTC NA H12 25 2 25 1 1 21 50

Results