Daniel:Notebook/ComboLock/2017-3-29: Difference between revisions

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>Djacobse
>Djacobse
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<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Add 1.5 uL reaction or ladder to appropriate aliquot</li>
<li>Add 1.5 uL reaction or ladder to appropriate aliquot</li>
[[Image:20170329-GelLanes-NEB.png|400px]]
[[Image:20170329-GelLanes-TF.png|400px]]
<li>Add 10 uL mix to gel lanes</li>
<li>Add 10 uL mix to gel lanes</li>
<li>Run gel for 24 minutes at 230V</li>
<li>Run gel for 24 minutes at 230V</li>
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Gel amounts might not be right. Too much ladder or too little sample
Gel amounts might not be right. Too much ladder or too little sample


==dsDNA Qubit==
==dsDNA Qubit==

Revision as of 18:46, 3 April 2017

RCA Test (Started March 24)

Back to Calendar

Bglii Digestion

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

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

dsDNA Qubit

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) 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