Daniel:Notebook/ComboLock/2016-10-6: Difference between revisions
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>Djacobse |
>Djacobse |
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==Protocol 2== | ==Protocol 2== | ||
<ol start="5"> | <ol start="5"> | ||
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<ol type="A"> | <ol type="A"> | ||
<li>Set up reaction according to table below</li> | <li>Set up reaction according to table below</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="45" | Sample | |||
| width="65" | Molecule Count | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Sample Vol (uL) | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM AmpF 6.4 | |||
| width="65" | 10 uM AmpR6.3-IndXX | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample1 | |||
| align="center" align="center" valign="bottom" | 1.00E+10 | |||
| align="center" valign="bottom" | A1 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample2 | |||
| align="center" align="center" valign="bottom" | 1.00E+10 | |||
| align="center" valign="bottom" | B1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample3 | |||
| align="center" align="center" valign="bottom" | 1.00E+10 | |||
| align="center" valign="bottom" | C1 | |||
| align="center" align="center" valign="bottom" | 26 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NTC | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" valign="bottom" | D1 | |||
| align="center" align="center" valign="bottom" | 27 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 19.5 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
===Results 2=== |
Revision as of 00:12, 7 October 2016
C Probe Positive Control Test (Started Yesterday)
Protocol
- 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
- qPCR
- Set up reaction according to table below
- Make master mixes according to following recipes
- 54.25 uL nfH2O
- 77.5 uL SYBR Fast
- 1.55 uL AmpF
- 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 x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 48 uL TBE, 12 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
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) |
Sample1 | 1.00E+10 | A1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample2 | 1.00E+10 | B1 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample3 | 1.00E+10 | C1 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | D1 | 27 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
Results
- 2016-10-06-qPCR-PCCTest1.png
qPCR curve
- 2016-10-06-PCCTest1-AmpFAmpR.png
Gel image
There is a faint band present at the correct size in the sample lane. I should recheck though by repeating the experiment above using more cycles.
Protocol 2
- qPCR
- Set up reaction according to table below
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) |
Sample1 | 1.00E+10 | A1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample2 | 1.00E+10 | B1 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample3 | 1.00E+10 | C1 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | D1 | 27 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |