Daniel:Notebook/ComboLock/2016-9-23: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Follow reaction map below</li> | |||
*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) | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| align="center" width="65" height="35" | | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 1 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 2 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 3 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 4 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 5 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 6 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 7 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 8 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 9 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 10 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 11 | |||
|style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 12 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | A | |||
| X,T1 | |||
| align="center" | | |||
| X,T2 | |||
| align="center" | | |||
| X,T3 | |||
| align="center" | | |||
| X,T4 | |||
| align="center" | | |||
| X,T5 | |||
| align="center" | | |||
| align="center" | | |||
| X,T6 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | B | |||
| X,T1 | |||
| align="center" | | |||
| X,T2 | |||
| align="center" | | |||
| X,T3 | |||
| align="center" | | |||
| X,T4 | |||
| align="center" | | |||
| X,T5 | |||
| align="center" | | |||
| align="center" | | |||
| X,T6 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | C | |||
| X,T1 | |||
| align="center" | | |||
| X,T2 | |||
| align="center" | | |||
| X,T3 | |||
| align="center" | | |||
| X,T4 | |||
| align="center" | | |||
| X,T5 | |||
| align="center" | | |||
| align="center" | | |||
| X,T6 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | D | |||
| O,T1 | |||
| align="center" | | |||
| O,T2 | |||
| align="center" | | |||
| O,T3 | |||
| align="center" | | |||
| O,T4 | |||
| align="center" | | |||
| O,T5 | |||
| align="center" | | |||
| align="center" | | |||
| O,T6 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | E | |||
| O,T1 | |||
| align="center" | | |||
| O,T2 | |||
| align="center" | | |||
| O,T3 | |||
| align="center" | | |||
| O,T4 | |||
| align="center" | | |||
| O,T5 | |||
| align="center" | | |||
| align="center" | | |||
| O,T6 | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | F | |||
| O,T1 | |||
| align="center" | | |||
| O,T2 | |||
| align="center" | | |||
| O,T3 | |||
| align="center" | | |||
| O,T4 | |||
| align="center" | | |||
| O,T5 | |||
| align="center" | | |||
| align="center" | | |||
| O,T6 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | G | |||
| NTC | |||
| align="center" | | |||
| NTC | |||
| align="center" | | |||
| NTC | |||
| align="center" | | |||
| NTC | |||
| align="center" | | |||
| NTC | |||
| align="center" | | |||
| align="center" | | |||
| NTC | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | H | |||
| align="center" | | |||
| align="center" | | |||
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| align="center" | | |||
| align="center" | | |||
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| align="center" | | |||
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| align="center" | | |||
|} | |||
<li>Set up reaction according to table</li> | <li>Set up reaction according to table</li> | ||
Revision as of 18:45, 23 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
Control
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
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- qPCR
- Set up reaction according to table
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- qPCR
- Set up reaction according to table
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- qPCR
- Set up reaction according to table
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
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 |
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 1A | 1.00E+10 | A1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 1.00E+10 | B1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 1.00E+10 | C1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2A | 1.00E+09 | D1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | 1.00E+09 | E1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2C | 1.00E+09 | F1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | A1 | 36 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Primer Concentration