Daniel:Notebook/ComboLock/2017-3-27

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RCA Test (Started March 24)[edit]

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Protocol[edit]

  1. Rolling Circle Amplification
    1. Add 20 uL nfH2O to every sample
  2. qPCR
    1. Make master mixes according to following table
    2. Reagent 1X Rxn MM-Amp (33.2X) MM-Prod (9.2X)
      Primer Pair NA AmpF/AmpR p4/p12RC
      Forward Primer (10 uM) 1 33.2 9.2
      Forward Primer (10 uM) 1 33.2 9.2
      Kapa SYBR Fast Master Mix (2X) 25 830 230
      nfH2O 21 697.2 193.2
      Total 48 1593.6 441.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170327-RCATest-CPLP.png
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  3. Qubit Measurement
    1. Add 995 uL buffer and 5 uL ssdye to tube
    2. Add 190 uL buffer and 10 uL correct standard to standard tubes
    3. Add 199 uL buffer and 1 uL sample to sample tubes
    4. Let sit 5-10 minutes in the dark
    5. Measure in Qubit
  4. TBE Gel
    1. Mix 120 uL TBE and 40 uL 6X dye
    2. Aliquot 10 uL per sample on to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to aliquots
    4. Mix and add 10 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc

Results[edit]

Qubit Results[edit]

Sample Name Condition ug/mL ng/mL bp nM
XA NEB RCA 3 hrs CP (LLRC) 4.27   188 68.8
XB NEB RCA 3 hrs CP (LLRC*) 24   188 386.9
XC NEB RCA 3 hrs CP (LLRC***) 37.4   188 602.8
XD NEB RCA 3 hrs LP (LLRC) 2.66   188 42.9
XE NEB RCA 3 hrs LP (LLRC*) 24.9   188 401.4
XF NEB RCA 3 hrs LP (LLRC***) 37.5   188 604.5
CP Circular Product Alone 12.1   188 195.0
PA NEB RCA 6 hrs CP (LLRC) 3.08   188 49.7
PB NEB RCA 6 hrs CP (LLRC*) 17.5   188 282.1
PC NEB RCA 6 hrs CP (LLRC***) 28.3   188 456.2
PD NEB RCA 6 hrs LP (LLRC) 1.84   188 29.7
PE NEB RCA 6 hrs LP (LLRC*) 17.9   188 288.5
PF NEB RCA 6 hrs LP (LLRC***) 29.5   188 475.5
LP Linear Product Alone   450 188 7.3
QA NEB RCA 12 hrs CP (LLRC) 4.87   188 78.5
QB NEB RCA 12 hrs CP (LLRC*) 20.2   188 325.6
QC NEB RCA 12 hrs CP (LLRC***) 29.6   188 477.1
QD NEB RCA 12 hrs LP (LLRC) 1.97   188 31.8
QE NEB RCA 12 hrs LP (LLRC*) 19.2   188 309.5
QF NEB RCA 12 hrs LP (LLRC***) 33.7   188 543.2


qPCR Results[edit]

Summary Results and Discussion[edit]

Based on the results here, I would say that the Thermo enzyme seemed to be the best (keep in mind Epicentre was mixed 1:10 before adding it to the thermocycler and is therefore not necessarily the best true comparison for Epi). Also, the amount of product does in fact amplify by time accordingly, that is for each different RCA primer (LLRC, LLRC*, and LLRC***) the amount of product present at 6 hours is approximately double the amount at 3 hours, and 12 hours is approximately double 6 hours. However, the baseline still appears to be off. That is, the total amount present should be much higher than is being elucidated in the qPCR at the moment.