Matt:LabNotes/2017-6-1: Difference between revisions
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>Mzcai |
>Mzcai (→FISH) |
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[[File:20170606_w2_composite.jpg|650px]]<br> | [[File:20170606_w2_composite.jpg|650px]]<br> | ||
===FISH=== | ===FISH=== | ||
*Olympus Epifluorescence | [[File:20170607_w1_composite.jpg|650px]] | ||
**Only saw signal in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads) | *Olympus Epifluorescence (GFP in green, Cy5 in red) | ||
**Only saw round signal that could be polony in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads) | |||
**No signal in Cy5 channel (tried exposure time from 100ms to 1s) | **No signal in Cy5 channel (tried exposure time from 100ms to 1s) | ||
*Magnetic beads were still on the gel, often much more clumped together | *Magnetic beads were still on the gel, often much more clumped together |
Latest revision as of 22:52, 6 June 2017
Image and Seq[edit]
http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm
Cast 10% gels with primer[edit]
- Gels were cast Matt:LabNotes/2017-5-24 and stored in water
Prepare Beads[edit]
- Prepare 50ml Dynabeads buffer: 20ml 5M NaCl + 100ul 0.5M EDTA + 125ul 4M Tris-HCl + 29.775ml H2O
- 2M NaCl, 1mM EDTA, 10mM Tris-HCl
- Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
- Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
- Vortex
- Pull down 1min on magnet
- Remove supernatant
- Combined 15ul buffer with 15ul amplicon
- BarcUv1 240nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
- BarcUv3 220nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
- Add to beads, pipette mix, and incubate 15min at room temp
- Pull down
- Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
- Bring volume to 40ul with H2O
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
- Saw polonies!
- Approx 8um in diameter and centered around the beads
- Confident not autofluorescence from the bead because only ~1/4 beads had a polony
- The 1/4 polony success rate might need to be improved but right now it may just be due to beads with no DNA
- Edit: might not be polony if SYBR Green stained dsDNA on the bead...
- Saw polonies!
- Put in 4C overnight
Strip Second Strand[edit]
- Remove SecureSeal and put slide in glass Coplin jar of Hexane for 5min
- Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
- Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100
- Shake slide in plastic box of Wash 1E twice for 4min
- Apply blue FrameSeal chamber
- Hoping to use smaller volumes of Wash 1E
- Add ~800ul Wash 1E and then aspirate 2 times
Strip Second Strand[edit]
- Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
- Wash twice with Wash 1E
Hybridize FISH probes[edit]
- Add 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC preheated to 80C and incubate 10min at room temp
- Wash twice with 2X SSC
- Add 2X SSC and then seal with glass coverslip
Results[edit]
SYBR Green[edit]
File:20170604 w2 composite.jpg
File:20170605 w2 composite.jpg
File:20170606 w2 composite.jpg
FISH[edit]
File:20170607 w1 composite.jpg
- Olympus Epifluorescence (GFP in green, Cy5 in red)
- Only saw round signal that could be polony in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads)
- No signal in Cy5 channel (tried exposure time from 100ms to 1s)
- Magnetic beads were still on the gel, often much more clumped together
- No signal under confocal
Conclusion[edit]
- Initially thought the SYBR Green images looked good
- The fluorescence signal was centered around beads and had diameter of ~8um after 15 cycles
- Only a fraction of beads had fluorescence suggesting it was not autofluorescence and true polonies
- Now after cleaning up fluorescent image and overlaying, it seems the fluorescence could be from the bead (maybe SYBR green binding to dsDNA on the bead)
- Fluorescent polony size and BF bead size seem to match perfectly
- No polony signal after FISH, could be due to bad FISH protocol or no polonies...
- Areas to improve:
- Cleaner gel (there is too much crap on the gel)
- Having a little debris is good for finding focus if there aren't any beads around
- Image SYBR Green with and without beads
- Image right after thermalcycler and then remove beads with magnet and image SYBR Green again
- Image after varying number of PCR cycles
- 1 cycle, 15 cycles, 30 cycles
- To improve FISH, follow the Harvard Protocol more closely
- Cleaner gel (there is too much crap on the gel)
Harvard Protocol[edit]
- Prepare 70% formamide 1X SSC in plastic coplin jar
- 4mL 20X SSC
- 56mL formamide
- 20mL H2O
- Heat to 70C in microwave (~30sec in microwave) being careful not to let it boil over
- Put slides in and shake in 70C incubator 15min
- Transfer slides to separate coplin jar
- Wash in dH2O 3min on shaker
- Wash twice in Wash 1E 4min on shaker