Daniel:Notebook/ComboLock/2016-10-6: Difference between revisions
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File:2016-10-06-PCCTest2-AmpFAmpR.png|Gel image | File:2016-10-06-PCCTest2-AmpFAmpR.png|Gel image | ||
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Since I let the run go a few more cycles, the signal is now prominent. However, there is significant AmpF amplification again. In fact, the sample lane was actually the slowest to amplify. So that just reiterates that I need to get rid of AmpF in the padlock. This is reflected in Padlock0301. | |||
[[Category:ComboLock]] [[Category:20161005]] |
Latest revision as of 18:43, 7 October 2016
C Probe Positive Control Test (Started Yesterday)[edit]
Protocol[edit]
- 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[edit]
- 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[edit]
- 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 2[edit]
- 2016-10-06-qPCR-PCCTest2.png
qPCR curve
- 2016-10-06-PCCTest2-AmpFAmpR.png
Gel image
Since I let the run go a few more cycles, the signal is now prominent. However, there is significant AmpF amplification again. In fact, the sample lane was actually the slowest to amplify. So that just reiterates that I need to get rid of AmpF in the padlock. This is reflected in Padlock0301.