Daniel:Notebook/ComboLock/2017-3-23: Difference between revisions

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===Results===
===Results===


<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=3 heights=300px widths=300px>
File:20170321-qPCR-T4Circ-PostBglii.png|qPCR curves
File:20170323-qPCR-CPProd.png|qPCR curves
File:2017-03-21-T4Circ-PostBglii-T4Samples.png|Gel image-T4 Ligase samples (F)
File:2017-03-23-CPProd-qPCR-CPTarget.png|Circular Product target (104bp)
File:2017-03-21-T4Circ-PostBglii-APSamples.png|Gel image-Amp/Phusion samples (G,H)
File:2017-03-23-CPProd-qPCR-AmpF-AmpR.png|AmpF/AmpR (221 bp)
</gallery>
</gallery>
Use the AmpF/AmpR gel as a reference point in the future. Also, positive controls for qPCR and the like should be done at a 1:10 dilution of the original. The original concentration, via Qubit is:
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#80FF00;font-size:12pt;font-weight:bold" align="center"
| width="85" height="30" | Qubit
| width="85" | Dil
| width="105" | Meas
| width="105" | Amt
| width="105" | Average (ng/uL)
| width="105" | uM
|- style="font-size:12pt"
| height="15"  valign="bottom" | x1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 118
| align="center" align="center" valign="bottom" | 118
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | x2
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 18.3
| align="center" align="center" valign="bottom" | 91.5
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
|- style="font-size:12pt"
| height="15"  valign="bottom" | x3
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 8.94
| align="center" align="center" valign="bottom" | 89.4
| align="center" align="center" valign="bottom" | 99.6
| align="center" align="center" valign="bottom" | 1.61
|}
=Linear Product Production=
This is the next step in planning my RCA experiment. This will serve as a negative control for the experiment, since no circular product should be formed.
==Protocol==
<ol>
<li>Dephosphorylation (Bolt Oligo)</li>
<ol type="A">
<li>Mix the following reaction</li>
<ol type="a">
<li>4 uL Circ Latch oligo (100 uM)</li>
<li>2 uL CutSmart Buffer (10X)</li>
<li>2 uL rSAP enzyme</li>
<li>12 uL  nfH2O</li></ol>
<li>Incubate at 37C for 30 minutes</li>
<li>Incubate at 65 for 5 minutes</li></ol>
<li>Ligation</li>
<ol type="A">
<li>Make reaction mixture according to table; '''Do not add ligase yet'''</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#66FFFF;font-size:12pt;font-weight:bold" align="center"
| width="220" height="36" | Reagent
| width="100" | Stock Conc
| width="100" | Final Conc./Amount
| width="106" | uL added
|- style="font-size:12pt"
| height="15"  valign="bottom" | T4 Ligase Buffer
| align="center" | 5X
| align="center" | 1X
| align="center" align="center" | 4
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Dephosphorylated Circ Latch (Step 1)
| align="center" | 20 uM
| align="center" | 100 pmol
| align="center" align="center" | 5
|- style="font-size:12pt"
| height="15"  valign="bottom" | Padlock 0601 oligo
| align="center" | 100 uM
| align="center" | 100 pmol
| align="center" align="center" | 1
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | C4 Positive Control oligo
| align="center" | 100 uM
| align="center" | 100 pmol
| align="center" align="center" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | C2 positive Control oligo
| align="center" | 100 uM
| align="center" | 100 pmol
| align="center" align="center" | 1
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | T4 DNA Ligase
| align="center" | 5 U/uL
| align="center" | 5 U
| align="center" align="center" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 6
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
|style="font-weight:bold" align="center" align="center" | 20
|}
<li>Use following thermocycler program</li>
<ol type="a">
<li>Heat mixture to 95C for 5 min</li>
<li>Lower temp by 0.2C per second to 55C</li>
<li>Hold mixture at 55C for 15 minutes</li>
<li>Lower temp to 20C by reducing 0.2C per second</li>
<li>Hold at 20C</li></ol>
<li>Add 1 uL T4 DNA ligase</li>
<li>Incubate at 20C for 30 minutes</li>
<li>Heat kill enzyme by incubating at 65C for 15 minutes</li>
<li>Add 20 uL nfH2O</li></ol>
<li>TBE gel</li>
<li>Mix 64 uL TBE and 16 uL 6X dye</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li>
[[Image:G|400px]]
<li>Add 10 uL mix to gel lanes</li>
<li>Run gel for 24 minutes at 230V</li>
<li>Stain with 2 uL SYBR gold for 3 minutes</li>
<li>Rinse and image in gel doc</li>
</ol>
[[Image:2017-03-24-LinearProductProduction.png|500px]]
[[Category:ComboLock]] [[Category:20170322]]

Latest revision as of 16:28, 24 March 2017

Circular Product Production (Started Yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. qPCR
    1. Make qPCR master mix according to following recipes
    2. Reagent 1X Rxn MM-Amp (6.2X) MM-Prod (7.2X)
      Primer Pair NA AmpF/AmpR p24/p12RC
      Forward Primer (10 uM) 1 6.2 7.2
      Forward Primer (10 uM) 1 6.2 7.2
      Kapa SYBR Fast Master Mix (2X) 25 155 180
      nfH2O 21 130.2 151.2
      Total 48 297.6 345.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170323-CPProduction.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
  2. 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
  3. 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]

Use the AmpF/AmpR gel as a reference point in the future. Also, positive controls for qPCR and the like should be done at a 1:10 dilution of the original. The original concentration, via Qubit is:

Qubit Dil Meas Amt Average (ng/uL) uM
x1 1 118 118    
x2 5 18.3 91.5    
x3 10 8.94 89.4 99.6 1.61

Linear Product Production[edit]

This is the next step in planning my RCA experiment. This will serve as a negative control for the experiment, since no circular product should be formed.

Protocol[edit]

  1. Dephosphorylation (Bolt Oligo)
    1. Mix the following reaction
      1. 4 uL Circ Latch oligo (100 uM)
      2. 2 uL CutSmart Buffer (10X)
      3. 2 uL rSAP enzyme
      4. 12 uL nfH2O
    2. Incubate at 37C for 30 minutes
    3. Incubate at 65 for 5 minutes
  2. Ligation
    1. Make reaction mixture according to table; Do not add ligase yet
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Ligase Buffer 5X 1X 4
      Dephosphorylated Circ Latch (Step 1) 20 uM 100 pmol 5
      Padlock 0601 oligo 100 uM 100 pmol 1
      C4 Positive Control oligo 100 uM 100 pmol 1
      C2 positive Control oligo 100 uM 100 pmol 1
      T4 DNA Ligase 5 U/uL 5 U 1
      nfH2O NA NA 6
      Total     20
    3. Use following thermocycler program
      1. Heat mixture to 95C for 5 min
      2. Lower temp by 0.2C per second to 55C
      3. Hold mixture at 55C for 15 minutes
      4. Lower temp to 20C by reducing 0.2C per second
      5. Hold at 20C
    4. Add 1 uL T4 DNA ligase
    5. Incubate at 20C for 30 minutes
    6. Heat kill enzyme by incubating at 65C for 15 minutes
    7. Add 20 uL nfH2O
  3. TBE gel
  4. Mix 64 uL TBE and 16 uL 6X dye
  5. Aliquot 10 uL on to parafilm per sample/ladder
  6. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
  7. File:G
  8. Add 10 uL mix to gel lanes
  9. Run gel for 24 minutes at 230V
  10. Stain with 2 uL SYBR gold for 3 minutes
  11. Rinse and image in gel doc

File:2017-03-24-LinearProductProduction.png