Daniel:Notebook/PosSequencing/2017-6-3: Difference between revisions
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>Djacobse (Created page with "=Aqueous PCR Test (Started Yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Wash St...") |
>Djacobse |
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</ol> | </ol> | ||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | |||
<li>Make the following 14.2X master mix (1X)</li> | <li>Make the following 14.2X master mix (1X)</li> | ||
<ol type="a"> | <ol type="a"> | ||
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<li>Add 2 uL sample according to plate layout below</li> | <li>Add 2 uL sample according to plate layout below</li> | ||
[[Image:|522x348px]] | [[Image:PlateLayout-20170603-aqPCR.png|522x348px]] | ||
<li>Run the following thermocycler program</li> | <li>Run the following thermocycler program</li> | ||
Line 42: | Line 43: | ||
</ol></ol> | </ol></ol> | ||
</ol> | </ol> | ||
==Results== | |||
<gallery perrow=2 heights=300px widths=400px mode=packed-hover> | |||
File:PlateLayout-20170603-aqPCR.png|Plate layout | |||
File:20170603-aqPCR-Fractions-plateCTs.png|Plate CT values | |||
File:20170603-aqPCR-Fractions-norm-plateCTs.png|Plate CT values-normalized | |||
File:20170603-aqPCR-Fractions-rawcurves.png|Raw curves | |||
</gallery> | |||
Conclusions: | |||
*All the 1:4 ratio samples peak 1-3 cycles behind the full sample. Makes sense given that it should be 2 | |||
*The H3 and H4 samples have too high of concentration. The H4 amplified in basically 0 samples and the H3 sample amplified even quicker, but the baseline correction really hurts the detection | |||
*There is basically nothing in any of the samples (at least above NTC baseline), but that unfortunately includes the elution fraction as well. It may require a 95C cook to denature them properly (technically 95C steps are in the qPCR also, but to elute I often use 10-15 minutes at 95C instead of like 3 | |||
[[Category:PosSeq]] [[Category:20170602]] |
Latest revision as of 18:28, 9 June 2017
Aqueous PCR Test (Started Yesterday)[edit]
Protocol[edit]
- Wash Steps
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: A)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: B)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: C)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: D)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix; Keep this fraction (Fraction: E)
- qPCR
- Make the following 14.2X master mix (1X)
- 14.2 uL 10 uM BiotinUracilPrimer1 (1)
- 14.2 uL 10 uM Primer2 (1)
- 284 uL 2X Kap SYBR Master Mix (20)
- 227.2 uL nfH2O (16)
- Aliquot 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-20170603-aqPCR.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x120
- 72C 2 min
- 16C hold
Results[edit]
- PlateLayout-20170603-aqPCR.png
Plate layout
- 20170603-aqPCR-Fractions-plateCTs.png
Plate CT values
- 20170603-aqPCR-Fractions-norm-plateCTs.png
Plate CT values-normalized
- 20170603-aqPCR-Fractions-rawcurves.png
Raw curves
Conclusions:
- All the 1:4 ratio samples peak 1-3 cycles behind the full sample. Makes sense given that it should be 2
- The H3 and H4 samples have too high of concentration. The H4 amplified in basically 0 samples and the H3 sample amplified even quicker, but the baseline correction really hurts the detection
- There is basically nothing in any of the samples (at least above NTC baseline), but that unfortunately includes the elution fraction as well. It may require a 95C cook to denature them properly (technically 95C steps are in the qPCR also, but to elute I often use 10-15 minutes at 95C instead of like 3