Daniel:Notebook/ComboLock/2017-2-3: Difference between revisions

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>Djacobse
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<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=2 heights=300px widths=300px>
File:|qPCR Curve
File:|qPCR Curve
File:|Gel Image
File:|Gel Image-PreRCA
File:2017-02-03-v2Oligos-Round2-PostRCA.png|Gel Image-PostRCA
File:|Gel Image-Epicentre
</gallery>
</gallery>


[[Category:ComboLock]] [[Category:20170202]]
[[Category:ComboLock]] [[Category:20170202]]

Revision as of 23:34, 3 February 2017

Version 2 Oligos Round 2 (Started yesterday)

Back to Calendar

Protocol-Part 4-RCA

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 8.2X Master mix according to table below; Give A samples LLRC and B Samples LLRC*
    2. Reagent uL Added Master Mix (8.2X) Master Mix (4.2X)
      Template 5 NA NA
      RCA Primer (10 uM) 2.5 0 0
      dNTP (1 mM) 5 41 21
      10X Buffer 2 16.4 8.4
      Phi29 1 0 0
      BSA (10 mg/mL) 0.4 3.28 0
      nfH2O 4.1 33.62 18.9
      Total 20 114.8 58.8
    3. Add 11.5 uL master mix to each tube
    4. Add sample and 1 uL appropriate enzyme to new reaction tubes
    5. Sample 1X (AB) 2X (AB) 3X (AB) 4X (AB) 1Y (AB) 4Y (AB)
      Phi29 Source NEB NEB NEB NEB Epicentre Epicentre
    6. Incubate at 37C for 3 hours
    7. Incubate at 65C for 10 minutes;Hold at 10C until next step

Protocol Part 5-qPCR and Gel

  1. qPCR
    1. Make 21.2X qPCR master mix according to following recipe
      1. 445.2 uL nfH2O
      2. 530 uL SYBR Fast
      3. 21.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
    4. Sample Condition Pre/Post RCA 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 Sample Pre A1 20 2 25 1 1 21 50
      Sample 1B Sample Pre A2 20 2 25 1 1 21 50
      Sample 2A No Template Pre A3 21 2 25 1 1 21 50
      Sample 2B No Template Pre A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Pre A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Pre A6 22 2 25 1 1 21 50
      Sample 4A No Lock Phosphate Pre A7 23 2 25 1 1 21 50
      Sample 4B No Lock Phosphate Pre A8 23 2 25 1 1 21 50
      Sample 1AX Sample Post H1 24 2 25 1 1 21 50
      Sample 1BX Sample Post H2 24 2 25 1 1 21 50
      Sample 2AX No Template Post H3 25 2 25 1 1 21 50
      Sample 2BX No Template Post H4 25 2 25 1 1 21 50
      Sample 3AX No C Probe Post H5 26 2 25 1 1 21 50
      Sample 3BX No C Probe Post H6 26 2 25 1 1 21 50
      Sample 4AX No Lock Phosphate Post H7 27 2 25 1 1 21 50
      Sample 4BX No Lock Phosphate Post H8 27 2 25 1 1 21 50
      Sample 1AY Sample Post D1 28 2 25 1 1 21 50
      Sample 1BY Sample Post D2 28 2 25 1 1 21 50
      Sample 4AY No Lock Phosphate Post D3 29 2 25 1 1 21 50
      Sample 4BY No Lock Phosphate Post D4 29 2 25 1 1 21 50
      NTC qPCR Primers Only   D5 30 2 25 1 1 23 50
    5. 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
  2. TBE Gel
    1. Mix 224 uL TBE, 56 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 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results