Daniel:Notebook/ComboLock/2016-8-28: Difference between revisions

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>Djacobse
(Created page with "=Positive Control Dilution Test (Started Friday)= Back to Calendar ==Protocol== <ol start="2"> <li>Cir...")
 
>Djacobse
 
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<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="105" height="45" | Sample
| width="78" | Molecule Count
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | Sample Vol (uL)
| 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 0
| align="center"  valign="bottom" | 1.00E+11
| align="center" valign="bottom" | A1
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 1
| align="center"  valign="bottom" | 1.00E+10
| align="center" valign="bottom" | B1
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 2
| align="center"  valign="bottom" | 1.00E+09
| align="center" valign="bottom" | C1
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 3
| align="center"  valign="bottom" | 1.00E+08
| align="center" valign="bottom" | D1
| align="center" align="center" valign="bottom" | 37
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 4
| align="center"  valign="bottom" | 1.00E+07
| align="center" valign="bottom" | E1
| align="center" align="center" valign="bottom" | 38
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 5
| align="center"  valign="bottom" | 1.00E+06
| align="center" valign="bottom" | F1
| align="center" align="center" valign="bottom" | 39
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 6
| align="center"  valign="bottom" | 100000
| align="center" valign="bottom" | G1
| align="center" align="center" valign="bottom" | 41
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 7
| align="center"  valign="bottom" | 10000
| align="center" valign="bottom" | H1
| align="center" align="center" valign="bottom" | 42
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 8
| align="center"  valign="bottom" | 1000
| align="center" valign="bottom" | A12
| align="center" align="center" valign="bottom" | 43
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 9
| align="center"  valign="bottom" | 100
| align="center" valign="bottom" | B12
| align="center" align="center" valign="bottom" | 44
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 10
| align="center"  valign="bottom" | 10
| align="center" valign="bottom" | C12
| align="center" align="center" valign="bottom" | 45
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 11
| align="center"  valign="bottom" | 1
| align="center" valign="bottom" | D12
| align="center" align="center" valign="bottom" | 46
| align="center" align="center" valign="bottom" | 2
| 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" | 17.5
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 12
| align="center"  valign="bottom" | 0
| align="center" valign="bottom" | E12
| align="center" align="center" valign="bottom" | 47
| align="center" align="center" valign="bottom" | 2
| 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" | 19.5
| align="center" align="center" valign="bottom" | 50
|}


<li>Master Mix</li>
<li>Master Mix</li>
Line 35: Line 206:
<li>plate read</li>
<li>plate read</li>
<li>goto b x20</li>
<li>goto b x20</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol></ol>
<li>qPCR Standard</li>
<ol type="A">
<li>Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
<li>Master Mix</li>
<ol type="a">
<li>352.5 uL KAPA SyberFast MM</li>
<li>310.2 uL nfH2O</li>
<li>7.05 uL primer2</li>
<li>7.05 uL primer12RC</li></ol>
<li>Add 48 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 x25</li>
<li>72C 2 min</li>
<li>72C 2 min</li>
<li>16C hold</li>
<li>16C hold</li>
</ol>
</ol>
</ol>
 
===Results===


<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=2 heights=300px widths=300px>
File:|X2 qPCR Curve
File:20160828-X2Dilution-qPCR.png|X2 Dilutions qPCR Curve
File:|X2 Gel Image
File:20160828-X2Dilution-Standard-qPCR.png|X2 Dilutions Standard qPCR Curve
File:2016-08-28-X2Dilution-0-5.png|Samples 0-5
File:2016-08-28-X2Dilution-6-11.png|Samples 0-5
File:20160829-X2Dilution-Primers-qPCR.png|Primer Amplification of Sample Amplicons; [[Daniel:Notebook/ComboLock/2016-8-29|08/29/2016]]
</gallery>
</gallery>
[[Category:ComboLock]] [[Category:20160826]]

Latest revision as of 20:05, 29 August 2016

Positive Control Dilution Test (Started Friday)[edit]

Back to Calendar

Protocol[edit]

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table
    2. Sample Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 0 1.00E+11 A1 34 2 25 0.5 5 17.5 50
      Sample 1 1.00E+10 B1 35 2 25 0.5 5 17.5 50
      Sample 2 1.00E+09 C1 36 2 25 0.5 5 17.5 50
      Sample 3 1.00E+08 D1 37 2 25 0.5 5 17.5 50
      Sample 4 1.00E+07 E1 38 2 25 0.5 5 17.5 50
      Sample 5 1.00E+06 F1 39 2 25 0.5 5 17.5 50
      Sample 6 100000 G1 41 2 25 0.5 5 17.5 50
      Sample 7 10000 H1 42 2 25 0.5 5 17.5 50
      Sample 8 1000 A12 43 2 25 0.5 5 17.5 50
      Sample 9 100 B12 44 2 25 0.5 5 17.5 50
      Sample 10 10 C12 45 2 25 0.5 5 17.5 50
      Sample 11 1 D12 46 2 25 0.5 5 17.5 50
      Sample 12 0 E12 47 2 25 0.5 5 19.5 50
    3. Master Mix
      1. 327.5 uL KAPA SyberFast MM
      2. 229.25 uL nfH2O
      3. 6.55 uL AmpF 6.4
    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
  4. qPCR Standard
    1. Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
    2. Master Mix
      1. 352.5 uL KAPA SyberFast MM
      2. 310.2 uL nfH2O
      3. 7.05 uL primer2
      4. 7.05 uL primer12RC
    3. Add 48 uL Master Mix to each well
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x25
      7. 72C 2 min
      8. 16C hold

      Results[edit]