Daniel:Notebook/ComboLock/2017-3-29

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RCA Test (Started March 24)[edit]

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Bglii Digestion[edit]

Testing bglii digestion of RCA product. No qPCR to start with (RCA should be enough). For recall, here is the sample matrix again and the RCA conditions used.

Sample Enzyme Incubation Time (hrs)
X (A-F) NEB 3
Y (A-F) Thermo 3
Z (A-F) Epi (1:10) 3
P (A-F) NEB 6
Q (A-F) NEB 12
  1. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (30.2X)
      RCA Rxn 2 0
      Bglii Cutter Primer + (100 uM) 2 60.4
      10X Buffer 3.1 2 60.4
      Bglii 1 0
      nfH2O 13 392.6
      Total 20 513.4
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 2 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min
  2. TBE gel
    1. Mix 200 uL TBE and 50 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 1.5 uL reaction or ladder to appropriate aliquot
    4. File:20170329-GelLanes-NEB.png File:20170329-GelLanes-TF.png
    5. Add 10 uL mix to gel lanes
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. Rinse and image in gel doc

Results[edit]

Gel amounts might not be right. Too much ladder or too little sample

dsDNA Qubit[edit]

Gonna try a double stranded DNA measurement in Qubit to see if there is any signal.

  1. Annealing
    1. Mix two uL reaction, 2 uL 100 uM Bglii + primer, and 6 uL nfH2O for samples XA-XF (NEB 3hr); include one XA with 2 uL 100 uM Bglii - primer
    2. Incubate at 95C for 5 min
    3. Incubate at 50C for 10 minutes
  2. Qubit
    1. Mix 1990 uL Qubit dsDNA buffer and 10 uL Qubit dsDNA dye
    2. Add 190 uL mix and 10 uL sample for S1 and S2 standards
    3. Add 199 uL mix and 1 uL sample for samples; include XA without bglii primer (8 samples total)
    4. Incubate in dark 5 minutes
    5. Measure in Qubit; use new calibration
    Sample Name Condition ug/mL ng/mL bp nM
    XA NEB RCA 3 hrs CP (LLRC) 45.5   188 733.4
    XB NEB RCA 3 hrs CP (LLRC*) 64   188 1031.59
    XC NEB RCA 3 hrs CP (LLRC***) 74.7   188 1204.06
    XD NEB RCA 3 hrs LP (LLRC) 65.1   188 1049.32
    XE NEB RCA 3 hrs LP (LLRC*) 47.7   188 768.86
    XF NEB RCA 3 hrs LP (LLRC***) 54.3   188 875.24
    XA (-) NEB RCA 3 hrs CP (LLRC) - Primer for antisense (-) strand 49.2   188 793.04
    XA-No primer NEB RCA 3 hrs CP (LLRC) No added primer 2.73   188 44