Daniel:Notebook/ComboLock/2016-7-29: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Phusion Test (Started yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Circularization</l...")
 
>Djacobse
 
(7 intermediate revisions by the same user not shown)
Line 20: Line 20:
| width="87" | Stock Concentration
| width="87" | Stock Concentration
| width="65" | Reagent Added
| width="65" | Reagent Added
| width="65" | uL Added per sample
| width="105" | uL Added per sample
| width="71" | Master Mix (5.1X)
| width="91" | Master Mix (5.1X)


|- style="font-size:12pt"  valign="bottom"
|- style="font-size:12pt"  valign="bottom"
Line 47: Line 47:
<ol type="A">
<ol type="A">
<li>Set up reaction according to table</li>
<li>Set up reaction according to table</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
| width="200" height="45" | Sample
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | Cells
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AmpF 6.4
| width="65" | 10 uM AmpR6.3-IndXX
| width="65" | H2O
| width="65" | Total Volume (uL)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 1 (Hs-Hemo Klentaq)
| align="center" valign="bottom" | A1
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 2 (Hs-Phusion)
| align="center" valign="bottom" | B1
| align="center" align="center" valign="bottom" | 16
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3 (Hs-No C-Probe)
| align="center" valign="bottom" | C1
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 4 (mm-Phusion)
| align="center" valign="bottom" | D1
| align="center" align="center" valign="bottom" | 18
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 5 (NTC)
| align="center" valign="bottom" | E1
| align="center" align="center" valign="bottom" | 19
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 6 (No Sample)
| align="center" valign="bottom" | F1
| align="center" align="center" valign="bottom" | 20
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 19
| align="center" align="center" valign="bottom" | 50
|}
<li>Master Mix</li>
<ol type="a">
<li>152.5 uL KAPA SyberFast MM</li>
<li>103.7 uL nfH2O</li>
<li>3.05 uL AmpF 6.4 100 uM</li></ol>
<li>Add 43 uL Master Mix to each well</li>
<li>qPCR Cycles</li>
<ol type="a">
<li>95C 3 min</li>
<li>95C 3 sec</li>
<li>55C 30 sec</li>
<li>72C 20 sec</li>
<li>plate read</li>
<li>goto b x20</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol>
</ol>
===qPCR Results===
<gallery perrow=2 heights=200px widths=200px>
File:2016-07-29-qPCR-PhusionTest.png|qPCR Curve
File:2016-07-29-PhusionTest.png|Gel Image
File:2016-07-29-PhusionTest-RawSample.png|Raw Sample Gel Image (TBU)
File:2016-07-29-PhusionTest-RawSample-Capture2.png|Raw Sample Gel Image (TBU)
</gallery>
[[Category:ComboLock]] [[Category:20160728]]

Latest revision as of 00:15, 18 August 2016

Phusion Test (Started yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
    2. Chill on ice 1 min
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 1 uL exonuclease III (100 units/μL) per sample
    2. Reagent Stock Concentration Reagent Added uL Added per sample Master Mix (5.1X)
      ExoI 20U/uL 20 1 5.1
      ExoIII 100 U/uL 100 1 5.1
    3. Add 2 μL of exonuclease I/III mix to reaction
    4. Mix the reaction by swirling pipette around the well 5 times
    5. Incubate reaction at 37 ºC for 2 hours
    6. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table
    2. Sample Lane AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 (Hs-Hemo Klentaq) A1 15 2.5 25 0.5 5 17 50
      Sample 2 (Hs-Phusion) B1 16 2.5 25 0.5 5 17 50
      Sample 3 (Hs-No C-Probe) C1 17 2.5 25 0.5 5 17 50
      Sample 4 (mm-Phusion) D1 18 2.5 25 0.5 5 17 50
      Sample 5 (NTC) E1 19 2.5 25 0.5 5 17 50
      Sample 6 (No Sample) F1 20 2.5 25 0.5 5 19 50
    3. Master Mix
      1. 152.5 uL KAPA SyberFast MM
      2. 103.7 uL nfH2O
      3. 3.05 uL AmpF 6.4 100 uM
    4. Add 43 uL Master Mix to each well
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold

    qPCR Results[edit]