Daniel:Notebook/ComboLock/2016-9-23: Difference between revisions

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=PCR Testing (Started [[Daniel:Notebook/ComboLock/2016-9-22|Yesterday]]=
=PCR Testing (Started [[Daniel:Notebook/ComboLock/2016-9-22|Yesterday]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]
Line 213: Line 213:
<li>plate read</li>
<li>plate read</li>
<li>goto b x24</li>
<li>goto b x24</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol></ol>
===Extension Time===
<ol>
<li>qPCR</li>
<ol type="A">
<li>Set up reaction according to table</li>
<li>Master Mix</li>
<ol type="a">
<li>480 uL KAPA SyberFast MM</li>
<li>336 uL nfH2O</li>
<li>9.5 uL AmpF 6.4</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>72C 2 min</li>
<li>16C hold</li>
<li>16C hold</li>
Line 246: Line 221:
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#948A54;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#948A54;font-size:12pt;font-weight:bold" align="center"
| width="69" height="45" | Sample
| width="105" height="45" | Sample
| width="65" | Molecule Count
| width="65" | Molecule Count
| width="65" | Lane
| width="65" | Lane
Line 274: Line 249:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
| 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" | A2
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 287: Line 262:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | C1
| align="center" valign="bottom" | A3
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 300: Line 275:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | D1
| align="center" valign="bottom" | A4
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 313: Line 288:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | E1
| align="center" valign="bottom" | A5
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 326: Line 301:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | F1
| align="center" valign="bottom" | A6
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 339: Line 314:
|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" | H1
| align="center" valign="bottom" | A7
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
Line 352: Line 327:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | A3
| align="center" valign="bottom" | B1
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 365: Line 340:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
| 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" | B2
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 378: Line 353:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | C3
| align="center" valign="bottom" | B3
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 391: Line 366:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | D3
| align="center" valign="bottom" | B4
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 404: Line 379:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | E3
| align="center" valign="bottom" | B5
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 417: Line 392:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | F3
| align="center" valign="bottom" | B6
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 430: Line 405:
|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" | H3
| align="center" valign="bottom" | B7
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
Line 443: Line 418:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | A5
| align="center" valign="bottom" | D1
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 456: Line 431:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | B5
| align="center" valign="bottom" | D2
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 469: Line 444:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" align="center" valign="bottom" | 1.00E+10
| align="center" valign="bottom" | C5
| align="center" valign="bottom" | D3
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 34
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 482: Line 457:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1A
| 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" | D4
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 495: Line 470:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1B
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | E5
| align="center" valign="bottom" | D5
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 508: Line 483:
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
|style="font-weight:bold" height="15"  valign="bottom" | Sample 1C
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" align="center" valign="bottom" | 1.00E+09
| align="center" valign="bottom" | F5
| align="center" valign="bottom" | D6
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 35
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 521: Line 496:
|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" | H5
| align="center" valign="bottom" | D7
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 36
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
Line 805: Line 780:


|}
|}
===Results===
The qPCR lanes are averages for sample lanes (not for NTCs)
<gallery perrow=3 heights=300px widths=300px>
File:2016-09-23_16-PosCon-65C.png|65C Annealing temp
File:2016-09-23_16-PosCon-64C.png|64C Annealing temp
File:2016-09-23_16-PosCon-59C.png|59C Annealing temp
File:2016-09-23_16-PosCon-55C.png|55C Annealing temp
File:2016-09-23_16-PosCon-51C.png|51C Annealing temp
File:2016-09-23_16-PosCon-50C.png|50C Annealing temp
File:20160923qPCR_SamplevsNTC.png|Samples (blue) vs NTC; different marker symbols indicate different temperatures
File:20160923qPCR_PrimerConc.png|Primer Concentration effects; 1 uM primers in light blue and 0.2 uM primer samples in dark blue
File:20160923qPCR_TempHiPrimer.png|Temperature dependence for 1 uM primer set
File:20160923qPCR_TempLoPrimer.png|Temperature dependence for 0.2 uM primer set
</gallery>
===Discussion===
Well, I'm not sure there is a clear indication, at least at the temperature range I used, that there is a temperature dependence. Comparing the 65C (theoretically the most stringent) and the 50C (least stringent) show little difference overall between the off-target bands. The lowered primer concentration does seem to help though, so I can try again on Monday with a continually decreasing level of primers. This also raises a point that the NTC consistently showed primer-dimer signal (~110bp). Looking back at some of the previous gels with NTCs ([[Daniel:Notebook/ComboLock/2016-8-19|this]] one shows it best), I think that the primer-dimers do form on their own but are helped by the presence of the padlock especially. Of note however is also that the number of cycles for this test was higher than usual. From previous PCR curves (see example from the [[Daniel:Notebook/ComboLock/2016-8-30|X3 dilution test]]) you can see that I went an extra 6 cycles or so and that the NTC lanes start to amplify around cycle 23.
[[Category:ComboLock]] [[Category:20160922]]

Latest revision as of 00:42, 28 September 2016

PCR Testing (Started Yesterday)[edit]

Back to Calendar

Protocol (all)[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


Standard qPCR like I normally do. Control version for the tests after.

  1. qPCR
    1. Follow reaction map below
      • Note:"X"=5 uL primer per reaction; "O" indicates 1 uL primer
      • Note:"TN" is a range of temperatures (gradient) from 50C (Lane 1) to 65C (Lane 12)
        1 2 3 4 5 6 7 8 9 10 11 12
      A X,T1   X,T2   X,T3   X,T4   X,T5     X,T6
      B X,T1   X,T2   X,T3   X,T4   X,T5     X,T6
      C X,T1   X,T2   X,T3   X,T4   X,T5     X,T6
      D O,T1   O,T2   O,T3   O,T4   O,T5     O,T6
      E O,T1   O,T2   O,T3   O,T4   O,T5     O,T6
      F O,T1   O,T2   O,T3   O,T4   O,T5     O,T6
      G NTC   NTC   NTC   NTC   NTC     NTC
      H                        
    2. Set up reaction according to table in next section
    3. Make master mixes according to following table
    4.   Master Mix
      Reagent MMX MMO NTC
      PrimerF (100 uM) 9.05 1.81 3.05
      PrimerR (10 uM) 90.5 18.1 30.5
      KAPA Master Mix 452.5 452.5 152.5
      nfH2O 316.75 396.39 118.95
    5. Add 48 uL correct master mix to each well
    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 x24
      7. 72C 2 min
      8. 16C hold

    qPCR Sample Table[edit]

    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) Master Mix
    Sample 1A 1.00E+10 A1 34 2 25 0.5 5 17.5 50 MMX
    Sample 1B 1.00E+10 A2 34 2 25 0.5 5 17.5 50 MMX
    Sample 1C 1.00E+10 A3 34 2 25 0.5 5 17.5 50 MMX
    Sample 1A 1.00E+09 A4 35 2 25 0.1 1 21.9 50 MMO
    Sample 1B 1.00E+09 A5 35 2 25 0.1 1 21.9 50 MMO
    Sample 1C 1.00E+09 A6 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 A7 36 0 25 0.5 5 19.5 50 NTC
    Sample 1A 1.00E+10 B1 34 2 25 0.5 5 17.5 50 MMX
    Sample 1B 1.00E+10 B2 34 2 25 0.5 5 17.5 50 MMX
    Sample 1C 1.00E+10 B3 34 2 25 0.5 5 17.5 50 MMX
    Sample 1A 1.00E+09 B4 35 2 25 0.1 1 21.9 50 MMO
    Sample 1B 1.00E+09 B5 35 2 25 0.1 1 21.9 50 MMO
    Sample 1C 1.00E+09 B6 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 B7 36 0 25 0.5 5 19.5 50 NTC
    Sample 1A 1.00E+10 D1 34 2 25 0.5 5 17.5 50 MMX
    Sample 1B 1.00E+10 D2 34 2 25 0.5 5 17.5 50 MMX
    Sample 1C 1.00E+10 D3 34 2 25 0.5 5 17.5 50 MMX
    Sample 1A 1.00E+09 D4 35 2 25 0.1 1 21.9 50 MMO
    Sample 1B 1.00E+09 D5 35 2 25 0.1 1 21.9 50 MMO
    Sample 1C 1.00E+09 D6 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 D7 36 0 25 0.5 5 19.5 50 NTC
    Sample 2A 1.00E+10 A7 34 2 25 0.5 5 17.5 50 MMX
    Sample 2B 1.00E+10 B7 34 2 25 0.5 5 17.5 50 MMX
    Sample 2C 1.00E+10 C7 34 2 25 0.5 5 17.5 50 MMX
    Sample 2A 1.00E+09 D7 35 2 25 0.1 1 21.9 50 MMO
    Sample 2B 1.00E+09 E7 35 2 25 0.1 1 21.9 50 MMO
    Sample 2C 1.00E+09 F7 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H7 36 0 25 0.5 5 19.5 50 NTC
    Sample 2A 1.00E+10 A9 34 2 25 0.5 5 17.5 50 MMX
    Sample 2B 1.00E+10 B9 34 2 25 0.5 5 17.5 50 MMX
    Sample 2C 1.00E+10 C9 34 2 25 0.5 5 17.5 50 MMX
    Sample 2A 1.00E+09 D9 35 2 25 0.1 1 21.9 50 MMO
    Sample 2B 1.00E+09 E9 35 2 25 0.1 1 21.9 50 MMO
    Sample 2C 1.00E+09 F9 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H9 36 0 25 0.5 5 19.5 50 NTC
    Sample 2A 1.00E+10 A12 34 2 25 0.5 5 17.5 50 MMX
    Sample 2B 1.00E+10 B12 34 2 25 0.5 5 17.5 50 MMX
    Sample 2C 1.00E+10 C12 34 2 25 0.5 5 17.5 50 MMX
    Sample 2A 1.00E+09 D12 35 2 25 0.1 1 21.9 50 MMO
    Sample 2B 1.00E+09 E12 35 2 25 0.1 1 21.9 50 MMO
    Sample 2C 1.00E+09 F12 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H12 36 0 25 0.5 5 19.5 50 NTC

    Results[edit]

    The qPCR lanes are averages for sample lanes (not for NTCs)

    Discussion[edit]

    Well, I'm not sure there is a clear indication, at least at the temperature range I used, that there is a temperature dependence. Comparing the 65C (theoretically the most stringent) and the 50C (least stringent) show little difference overall between the off-target bands. The lowered primer concentration does seem to help though, so I can try again on Monday with a continually decreasing level of primers. This also raises a point that the NTC consistently showed primer-dimer signal (~110bp). Looking back at some of the previous gels with NTCs (this one shows it best), I think that the primer-dimers do form on their own but are helped by the presence of the padlock especially. Of note however is also that the number of cycles for this test was higher than usual. From previous PCR curves (see example from the X3 dilution test) you can see that I went an extra 6 cycles or so and that the NTC lanes start to amplify around cycle 23.