Daniel:Notebook/ComboLock/2016-6-24: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> | <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> | ||
</ol> | </ol> | ||
<li>qPCR</li> | <li>qPCR *this is actually PCR since the eppendorf thermocycler was down and the other was in use</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up reaction according to table</li> | <li>Set up reaction according to table</li> | ||
Line 149: | Line 149: | ||
</ol> | </ol> | ||
</ol> | </ol> | ||
<li>PAGE gel</li> | |||
<ol type="A"> | |||
<li>Set up lanes according to table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#E6B8B7;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="35" | Sample | |||
| width="65" | Lane | |||
| width="65" | uL TBE Buffer | |||
| width="65" | uL loading dye | |||
| width="65" | uL sample | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Ladder | |||
| align="center" | 2 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Batch1 | |||
| align="center" | 3 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Batch2 | |||
| align="center" | 4 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Batch3 | |||
| align="center" | 5 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Batch4 | |||
| align="center" | 6 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Batch5 | |||
| align="center" | 7 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Batch6 | |||
| align="center" | 8 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Batch7 | |||
| align="center" | 9 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Batch8 | |||
| align="center" | 10 | |||
| align="center" | 4 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|} | |||
<li>Run gel for 25 minutes at 220V</li> | |||
</ol> | </ol> |
Revision as of 18:10, 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 *this is actually PCR since the eppendorf thermocycler was down and the other was in use
- 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
- PAGE gel
- Set up lanes according to table
- Run gel for 25 minutes at 220V
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 1 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 2 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 3 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 4 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 5 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | B6 | 6 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | A7 | B7 | 7 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | A8 | B8 | 8 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample | Lane | uL TBE Buffer | uL loading dye | uL sample |
Ladder | 2 | 4 | 2 | 2 |
Batch1 | 3 | 4 | 2 | 2 |
Batch2 | 4 | 4 | 2 | 2 |
Batch3 | 5 | 4 | 2 | 2 |
Batch4 | 6 | 4 | 2 | 2 |
Batch5 | 7 | 4 | 2 | 2 |
Batch6 | 8 | 4 | 2 | 2 |
Batch7 | 9 | 4 | 2 | 2 |
Batch8 | 10 | 4 | 2 | 2 |