Daniel:Notebook/ComboLock/2016-6-24: Difference between revisions
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>Djacobse |
>Djacobse |
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Line 26: | Line 26: | ||
| width="65" | Lane | | width="65" | Lane | ||
| width="65" | Batch | | width="65" | Batch | ||
| width="65" | AmpR Index | |||
| width="65" | Cells | | width="65" | Cells | ||
| width="65" | 2X Kapa SYBR qPCR MM | | width="65" | 2X Kapa SYBR qPCR MM | ||
| width="65" | 100 uM | | width="65" | 100 uM AmpF 6.4 | ||
| width="65" | | | width="65" | 10 uM AmpR-IndXX | ||
| width="65" | H2O | | width="65" | H2O | ||
| width="65" | Total Volume (uL) | | width="65" | Total Volume (uL) | ||
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| align="center" valign="bottom" | A1 | | align="center" valign="bottom" | A1 | ||
| align="center" valign="bottom" | B1 | | align="center" valign="bottom" | B1 | ||
| align="center" align="center" valign="bottom" | 71 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | 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" valign="bottom" | A2 | | align="center" valign="bottom" | A2 | ||
| align="center" valign="bottom" | B2 | | align="center" valign="bottom" | B2 | ||
| align="center" align="center" valign="bottom" | 72 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | 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" valign="bottom" | A3 | | align="center" valign="bottom" | A3 | ||
| align="center" valign="bottom" | B3 | | align="center" valign="bottom" | B3 | ||
| align="center" align="center" valign="bottom" | 73 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | 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" valign="bottom" | A4 | | align="center" valign="bottom" | A4 | ||
| align="center" valign="bottom" | B4 | | align="center" valign="bottom" | B4 | ||
| align="center" align="center" valign="bottom" | 74 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
Line 81: | Line 86: | ||
| align="center" valign="bottom" | A5 | | align="center" valign="bottom" | A5 | ||
| align="center" valign="bottom" | B5 | | align="center" valign="bottom" | B5 | ||
| align="center" align="center" valign="bottom" | 75 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
Line 92: | Line 98: | ||
| align="center" valign="bottom" | A6 | | align="center" valign="bottom" | A6 | ||
| align="center" valign="bottom" | B6 | | align="center" valign="bottom" | B6 | ||
| align="center" align="center" valign="bottom" | 76 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | 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" valign="bottom" | A7 | | align="center" valign="bottom" | A7 | ||
| align="center" valign="bottom" | B7 | | align="center" valign="bottom" | B7 | ||
| align="center" align="center" valign="bottom" | 77 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
Line 114: | Line 122: | ||
| align="center" valign="bottom" | A8 | | align="center" valign="bottom" | A8 | ||
| align="center" valign="bottom" | B8 | | align="center" valign="bottom" | B8 | ||
| align="center" align="center" valign="bottom" | 78 | |||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
|} | |} | ||
<li>Master Mix</li> | |||
<ol type="a"> | |||
<li>200 uL KAPA SyberFast MM</li> | |||
<li>140 uL nfH2O</li> | |||
<li>4 uL AmpF 6.4</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"> |
Revision as of 16:28, 24 June 2016
Stage 2 Test (Started yesterday)
Protocol
- Latch and Padlock Hybridization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 2 uL exonuclease I (20 units/μL) and 10 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
- qPCR
- Set up reaction according to table
- Master Mix
- 200 uL KAPA SyberFast MM
- 140 uL nfH2O
- 4 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
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 71 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 72 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 73 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 74 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 75 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | B6 | 76 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | A7 | B7 | 77 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | A8 | B8 | 78 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |