Daniel:Notebook/ComboLock/2016-9-23: Difference between revisions
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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|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" | | | 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 | ||
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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" | | | 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 |
Revision as of 18:13, 26 September 2016
PCR Testing (Started Yesterday)
Protocol (all)
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- 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
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
Standard qPCR like I normally do. Control version for the tests after.
- qPCR
- 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)
- Set up reaction according to table in next section
- Make master mixes according to following table
- Add 48 uL correct master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- 2016-09-23 16-PosCon-65C.png
65C Annealing temp
- 2016-09-23 16-PosCon-64C.png
64C Annealing temp
- 2016-09-23 16-PosCon-59C.png
59C Annealing temp
- 2016-09-23 16-PosCon-55C.png
55C Annealing temp
- 2016-09-23 16-PosCon-51C.png
51C Annealing temp
- 2016-09-23 16-PosCon-50C.png
50C Annealing temp
- 20160923qPCR SamplevsNTC.png
Samples (blue) vs NTC; different marker symbols indicate different temperatures
- 20160923qPCR PrimerConc.png
Primer Concentration effects; 1 uM primers in light blue and 0.2 uM primer samples in dark blue
- 20160923qPCR TempHiPrimer.png
Temperature dependence for 1 uM primer set
- 20160923qPCR TempLoPrimer.png
Temperature dependence for 0.2 uM primer set
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 |
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 |
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) | 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
The qPCR lanes are averages for sample lanes (not for NTCs)
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 (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.