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

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>Djacobse
>Djacobse
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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==
Gonna try a double stranded DNA measurement in Qubit to see if there is any signal.
<ol>
<li>Annealing</li>
<ol type="A">
<li>Mix two uL reaction, 2 uL 100 uM Bglii + primer, and 6 uL nfH2O for samples XA-XF (NEB 3hr)</li>
<li>Incubate at 95C for 5 min</li>
<li>Incubate at 50C for 10 minutes</li></ol>
<li>Qubit</li>
<ol type="A">
<li>Mix 1791 uL Qubit dsDNA buffer and 9 uL Qubit dsDNA dye</li>
<li>Add 190 uL mix and 10 uL sample for S1 and S2 standards</li>
<li>Add 199 uL mix and 1 uL sample for samples; include XA without bglii primer (7 samples total)</li>
<li>Incubate in dark 5 minutes</li>
<li>Measure in Qubit; use new calibration</li></ol> 
[[Category:ComboLock]] [[Category:20170324]]
[[Category:ComboLock]] [[Category:20170324]]

Revision as of 23:28, 29 March 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. 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

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)
    2. Incubate at 95C for 5 min
    3. Incubate at 50C for 10 minutes
  2. Qubit
    1. Mix 1791 uL Qubit dsDNA buffer and 9 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 (7 samples total)
    4. Incubate in dark 5 minutes
    5. Measure in Qubit; use new calibration