Daniel:Notebook/ComboLock/2016-12-14

From ZhangLabWiki
Revision as of 00:50, 15 December 2016 by >Djacobse (→‎Pre-RCA Beads vs Supernatant)
Jump to navigation Jump to search

Mirror Probe Test (Started Yesterday)

Back to Calendar

Protocol

  1. Exonuclease Digestion
    1. Mix 12.5 uL Exo I (20U/uL) and 2.5 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 7X Master mix according to table below
    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 (7X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 17.5
      dNTP (1 mM) 0.8 5.6
      10X Buffer 2 14
      Phi29 1 7
      nfH2O 8.7 60.9
      Total 20 105
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  3. qPCR
    1. Make master mix according to following recipe
      1. 151.2 uL nfH2O
      2. 180 uL SYBR Fast
      3. 7.2 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 in the following section
    4. Sample Condition 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 A1 20 2 25 1 1 21 50
      Sample 1B Sample A2 20 2 25 1 1 21 50
      Sample 2A No Template A3 21 2 25 1 1 21 50
      Sample 2B No Template A4 21 2 25 1 1 21 50
      Sample 3A No C Probe A5 22 2 25 1 1 21 50
      Sample 3B No C Probe A6 22 2 25 1 1 21 50
      NTC qPCR Primers Only A7 23 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
  4. TBE Gel
    1. Mix 80 uL TBE, 20 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

Results

So, nothing on this looks good. The PCR was more promising, with the sample lanes and (less encouragingly) N C Probe lanes showing a slightly higher CT than the No Template which was better than the NTC.

I'm going to try looking at the beads effect more closely now. I'll do PCR off the supernatant and beads fractions, as well as RCA on the beads fraction to test.

Beads vs Supernatant Fractions

I also might have neglected the dNTPs for RCA earlier. Just to make sure, I'm also doing a supernatant fraction RCA amplification. Protocol is the same for both.

  1. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    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 (7X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 17.5
      dNTP (1 mM) 0.8 5.6
      10X Buffer 2 14
      Phi29 1 7
      nfH2O 8.7 60.9
      Total 20 105
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

Continued tomorrow

Pre-RCA Beads vs Supernatant

  1. qPCR
    1. Make master mix according to following recipe
      1. 298.2 uL nfH2O
      2. 355 uL SYBR Fast
      3. 14.2 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 in the following section
    4. Sample Condition Fraction 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 Supernatant A1 20 2 25 1 1 21 50
      Sample 1B Sample Supernatant A2 20 2 25 1 1 21 50
      Sample 2A No Template Supernatant A3 21 2 25 1 1 21 50
      Sample 2B No Template Supernatant A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Supernatant A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Supernatant A6 22 2 25 1 1 21 50
      Sample 1A Sample Beads H1 23 2 25 1 1 21 50
      Sample 1B Sample Beads H2 23 2 25 1 1 21 50
      Sample 2A No Template Beads H3 24 2 25 1 1 21 50
      Sample 2B No Template Beads H4 24 2 25 1 1 21 50
      Sample 3A No C Probe Beads H5 25 2 25 1 1 21 50
      Sample 3B No C Probe Beads H6 25 2 25 1 1 21 50
      NTC qPCR Primers Only NA A7 26 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 80 uL TBE, 20 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

Results