Matt:LabNotes/2017-6-8
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Image and Seq[edit]
Plan[edit]
- PCR 3 gels and take them out at different cycles: 1, 15, and 30
Gel PCR[edit]
Prepare diffuse-in mix while allowing slides to dry. You don't want the slides to over-dry, though. You will observe a thin, shrinking film of liquid on the surface of each gel. Generally, we try to add the diffuse-in mix within 5 minutes or so of this shrinking film's disappearance. This generally means about 30 minutes of drying.
- Let gel slide dry in AirClean hood for 30min
- Prepare 25ul PCR mix per gel
- 2.5ul 5uM Primer2
- 12.5ul KAPA SYBR FAST MM
- 10ul beads (1:1 mix of v1 and v3)
- Pipet 25ul onto center of gel
- Apply 18x30mm cover slip
- Apply an orange SecureSeal chamber
- Fill chamber with mineral oil and seal holes with stickies.
- Slide PCR on Biorad thermocycler
94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
- Place directly on Olympus with GFP/FITC filter
Results[edit]
1 Cycle[edit]
- 1um spots but not near beads
- Beads can be seen drifting
File:20170608 1cycle.jpgFile:20170608 1cycle pos2.jpg
15 Cycles[edit]
- Almost no signal except for on a couple beads
File:20170608 15cycle.jpgFile:20170608 15cycle pos2.jpg
30 Cycles[edit]
- Large fraction (~50%) of beads have fluorescence
File:20170608 30cycle pos2.jpgFile:20170608 30cycle pos3.jpg
Conclusion[edit]
- I don't know what the signal was in 1cycle samples
- 15 and 30 cycles seems to have only amplified on beads
- Hypothesis: the high number of primers/template strand on beads outcompetes the acrydite primers in gel
- Dr. Zhang suggested adding 1/10th (50nM) of free floating Acrydite primer to help efficiency