Daniel:Notebook/ComboLock/2017-2-8

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Revision as of 00:02, 9 February 2017 by >Djacobse (→‎Results-Ethanol Precipitation)
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v3 Oligos-Round 3

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Protocol-Part 4

  1. qPCR
    1. Make 17.2X qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 430 uL SYBR Fast
      3. 17.2 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
      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 192 uL TBE, 48 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

Protocol-Part 5

  1. Ethanol Precipitation
    1. Centrifuge at 4C for 30 minutes at 12000 rpm
    2. Remove supernatant and add 750 uL cold 70%EtOH
    3. Centrifuge at 4C for 20 minutes at 12000 rpm
    4. Remove supernatant and vacuum centrifuge for 15 minutes to completely dry
    5. Resuspend pellet in 20 uL nfH2O
  1. Rolling Circle Amplification
    1. Prepare 8.2X Master mix according to table below; use LLRC primer for A samples and LLRC* (phosphorothiorate) samples for B samples
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.2X)
      Template 3 0
      RCA Primer (10 uM) 2.5 20.5
      dNTP (1 mM) 0.8 6.56
      10X Buffer 2 16.4
      Phi29 1 0
      nfH2O 10.7 87.74
      Total 20 131.2
    5. Incubate at 30C for 6 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

Protocol-Part 6

  1. qPCR
    1. Make 17.2X qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 430 uL SYBR Fast
      3. 17.2 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-EtOH+ Pre A1 20 2 25 1 1 21 50
      Sample 1B Sample-EtOH+ Pre A2 20 2 25 1 1 21 50
      Sample 2A No Template-EtOH+ Pre A3 21 2 25 1 1 21 50
      Sample 2B No Template-EtOH+ Pre A4 21 2 25 1 1 21 50
      Sample 3A No C Probe-EtOH+ Pre A5 22 2 25 1 1 21 50
      Sample 3B No C Probe-EtOH+ Pre A6 22 2 25 1 1 21 50
      Sample 4A No Lock Phosphate-EtOH+ Pre A7 23 2 25 1 1 21 50
      Sample 4B No Lock Phosphate-EtOH+ Pre A8 23 2 25 1 1 21 50
      Sample 1AX Sample-EtOH+ Post H1 24 2 25 1 1 21 50
      Sample 1BX Sample-EtOH+ Post H2 24 2 25 1 1 21 50
      Sample 2AX No Template-EtOH+ Post H3 25 2 25 1 1 21 50
      Sample 2BX No Template-EtOH+ Post H4 25 2 25 1 1 21 50
      Sample 3AX No C Probe-EtOH+ Post H5 26 2 25 1 1 21 50
      Sample 3BX No C Probe-EtOH+ Post H6 26 2 25 1 1 21 50
      Sample 4AX No Lock Phosphate-EtOH+ Post H7 27 2 25 1 1 21 50
      Sample 4BX No Lock Phosphate-EtOH+ Post H8 27 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 192 uL TBE, 48 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-Normal Protocol

As the highlighted band shows, sample A might actually have amplified since there is a faint band at the correct size that is not in the other lanes. However, it is faint and about equal to the incorrect band at 200bp.

Results-Ethanol Precipitation