Daniel:Notebook/ComboLock/2017-4-3

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RCA Test (Started March 24)[edit]

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I'll try qPCR on the RCA product again, this time on the NEB 12 hr samples (since I am running low on X samples), but for few cycles and with only the forward primer (which should be RC to the RCA product. This will be fed into Bglii digestion

  1. qPCR-Second strand synthesis
    1. Make the following 13.2X master mix
      1. 13.2 uL AmpF-6.4Sol (10 uM)
      2. 264 uL Kapa SYBR 2X Master Mix
      3. 224.4 uL nfH2O
    2. Aliquot 38 uL master mix into each lane
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-20170403-RCATest-CPLP-ssSynthesis.png
    5. Run the following protocol
      1. 95C for 2 min
      2. 95C for 5 sec
      3. 55C for 30 sec
      4. 72C for 45 sec
      5. Goto (b) x20
      6. 72C for 2 min

Protocol-Post-Bglii samples[edit]

  1. TBE Gel
    1. Mix 80 uL TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2.5 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. Run gel for 24 minutes at 230V
    6. Stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc
  2. Qiaquick Column
    1. Add 200 uL (5X) PB buffer to 40 uL sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    4. Dry spin column for 1 minute at 14000 rpm; discard flow through
    5. Let stand with cover open in fume hood for ~5 minutes
    6. Transfer column to a new 1.5 mL eppendorf tube
    7. Add 35 uL nfH2O to column
    8. Let stand 1 minute
    9. Spin for 1 minute at 14000 rpm
  3. TBE Gel
    1. Mix 40 uL 10X TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample, and 10 uL TBE ladder
    3. Add 6 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. Run gel for 24 minutes at 230V
    6. Stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc

Results[edit]

Purification really didn't do much but it the gels look good. The product should be around 200bp, btw, because I only used AmpF, which adds on about 20bp

Protocol-Pre-Bglii samples[edit]

  1. Bgl Digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (6.2X)
      RCA Rxn 8 0
      10X Buffer 3.1 2 12.4
      Bglii 1 0
      nfH2O 9 55.8
      Total 20 68.2
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 1 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min
  2. TBE Gel
    1. Mix 60 uL 10X TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
    3. Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. Run gel for 24 minutes at 230V
    6. Stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc

File:2017-04-04 -RCATest-CPLP-ssSynthesis-sssThenBgl.png

It appears that the Bgl digestion was incomplete. That does mean there is a lot more product available than can be cut in an hour with one uL of Bglii, which according to NEB is 10 ug (1 ug/unit/hr)