Daniel:Notebook/ComboLock/2016-10-1: Difference between revisions

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>Djacobse
>Djacobse
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<li>Make master mixes according to following recipes</li>
<li>Make master mixes according to following recipes</li>
<ol type="a">
<ol type="a">
 
<li>334.25 uL nfH2O</li>
 
<li>477.5 uL SYBR Fast</li>
 
<li>9.55 uL AmpF</li>
<li>Add 48 uL correct master mix to each well</li>
</ol>
<li>Add 43 uL master mix to each well</li>
<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
<ol type="a">
<ol type="a">
Line 36: Line 37:
<li>72C 2 min</li>
<li>72C 2 min</li>
<li>16C hold</li>
<li>16C hold</li>
</ol></ol>
</ol></ol></ol>


===qPCR Sample Table===
===qPCR Sample Table===
Line 98: Line 99:
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


Line 122: Line 123:
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | Sample 2A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2A
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | B1
| align="center" valign="bottom" | A7
| align="center" align="center" valign="bottom" | 26
| align="center" align="center" valign="bottom" | 26
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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|style="font-weight:bold" height="15"  valign="bottom" | Sample 2B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2B
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | B2
| align="center" valign="bottom" | A8
| align="center" align="center" valign="bottom" | 26
| align="center" align="center" valign="bottom" | 26
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 152: Line 153:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2C
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | B3
| align="center" valign="bottom" | H1
| align="center" align="center" valign="bottom" | 26
| align="center" align="center" valign="bottom" | 26
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 164: Line 165:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 3A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 3A
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | B4
| align="center" valign="bottom" | H2
| align="center" align="center" valign="bottom" | 27
| align="center" align="center" valign="bottom" | 27
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | Sample 3B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 3B
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | B5
| align="center" valign="bottom" | H3
| align="center" align="center" valign="bottom" | 27
| align="center" align="center" valign="bottom" | 27
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | Sample 3C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 3C
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | B6
| align="center" valign="bottom" | H4
| align="center" align="center" valign="bottom" | 27
| align="center" align="center" valign="bottom" | 27
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | Sample 4A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 4A
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | D1
| align="center" valign="bottom" | H5
| align="center" align="center" valign="bottom" | 28
| align="center" align="center" valign="bottom" | 28
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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|style="font-weight:bold" height="15"  valign="bottom" | Sample 4B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 4B
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | D2
| align="center" valign="bottom" | H6
| align="center" align="center" valign="bottom" | 28
| align="center" align="center" valign="bottom" | 28
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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|style="font-weight:bold" height="15"  valign="bottom" | Sample 4C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 4C
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | D3
| align="center" valign="bottom" | H7
| align="center" align="center" valign="bottom" | 28
| align="center" align="center" valign="bottom" | 28
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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|style="font-weight:bold" height="15"  valign="bottom" | Sample 5A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 5A
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | D4
| align="center" valign="bottom" | H8
| align="center" align="center" valign="bottom" | 29
| align="center" align="center" valign="bottom" | 29
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | Sample 5B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 5B
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | D5
| align="center" valign="bottom" | D1
| align="center" align="center" valign="bottom" | 29
| align="center" align="center" valign="bottom" | 29
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | Sample 5C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 5C
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | D6
| align="center" valign="bottom" | D2
| align="center" align="center" valign="bottom" | 29
| align="center" align="center" valign="bottom" | 29
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 21.9
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


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|style="font-weight:bold" height="15"  valign="bottom" | NTC
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" valign="bottom" | D7
| align="center" valign="bottom" | D3
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 30
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0

Revision as of 20:44, 1 October 2016

PCR Testing (Started Yesterday)

Back to Calendar

Protocol

  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 in next section
    2. Make master mixes according to following recipes
      1. 334.25 uL nfH2O
      2. 477.5 uL SYBR Fast
      3. 9.55 uL AmpF
    3. Add 43 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 x24
      7. 72C 2 min
      8. 16C hold

qPCR Sample Table

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 0A 1.00E+10 A1 24 2 25 0.5 5 17.5 50
Sample 0B 1.00E+10 A2 24 2 25 0.5 5 17.5 50
Sample 0C 1.00E+10 A3 24 2 25 0.5 5 17.5 50
Sample 1A 1.00E+09 A4 25 2 25 0.5 5 17.5 50
Sample 1B 1.00E+09 A5 25 2 25 0.5 5 17.5 50
Sample 1C 1.00E+09 A6 25 2 25 0.5 5 17.5 50
Sample 2A 1.00E+10 A7 26 2 25 0.5 5 17.5 50
Sample 2B 1.00E+10 A8 26 2 25 0.5 5 17.5 50
Sample 2C 1.00E+10 H1 26 2 25 0.5 5 17.5 50
Sample 3A 1.00E+09 H2 27 2 25 0.5 5 17.5 50
Sample 3B 1.00E+09 H3 27 2 25 0.5 5 17.5 50
Sample 3C 1.00E+09 H4 27 2 25 0.5 5 17.5 50
Sample 4A 1.00E+10 H5 28 2 25 0.5 5 17.5 50
Sample 4B 1.00E+10 H6 28 2 25 0.5 5 17.5 50
Sample 4C 1.00E+10 H7 28 2 25 0.5 5 17.5 50
Sample 5A 1.00E+09 H8 29 2 25 0.5 5 17.5 50
Sample 5B 1.00E+09 D1 29 2 25 0.5 5 17.5 50
Sample 5C 1.00E+09 D2 29 2 25 0.5 5 17.5 50
NTC 0 D3 30 0 25 0.5 5 19.5 50