Matt:LabNotes/2017-6-8: Difference between revisions

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>Mzcai
(Created page with "=Image and Seq= *Last time ==Plan== *PCR 3 gels and take them out at different cycles: 1, 15, and 30 ==Gel PCR== Prepare diffuse-in m...")
 
>Mzcai
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   94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
   94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold


#Place directly on Olympus with GFP/FITC filter<!--
#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...
#Put in 4C overnight
 
==Strip Second Strand==
#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==
#Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
#Wash twice with Wash 1E
 
==Hybridize FISH probes==
#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==
==Results==
===SYBR Green===
===1 Cycle===
[[File:20170604_w2_composite.jpg|650px]]<br>
*1um spots but not near beads
[[File:20170605_w2_composite.jpg|650px]]<br>
*Beads can be seen drifting
[[File:20170606_w2_composite.jpg|650px]]<br>
[[File:20170608_1cycle.jpg|450px]][[File:20170608_1cycle_pos2.jpg|450px]]
===FISH===
===15 Cycles===
[[File:20170607_w1_composite.jpg|650px]]
*Almost no signal except for on a couple beads
*Olympus Epifluorescence (GFP in green, Cy5 in red)
[[File:20170608_15cycle.jpg|450px]][[File:20170608_15cycle_pos2.jpg|450px]]
**Only saw round signal that could be polony in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads)
===30 Cycles===
**No signal in Cy5 channel (tried exposure time from 100ms to 1s)
*Large fraction (~50%) of beads have fluorescence
*Magnetic beads were still on the gel, often much more clumped together
[[File:20170608_30cycle_pos2.jpg|450px]][[File:20170608_30cycle_pos3.jpg|450px]]
*No signal under confocal


==Conclusion==
==Conclusion==
*Initially thought the SYBR Green images looked good
*I don't know what the signal was in 1cycle samples
**The fluorescence signal was centered around beads and had diameter of ~8um after 15 cycles
*15 and 30 cycles seems to have only amplified on beads
**Only a fraction of beads had fluorescence suggesting it was not autofluorescence and true polonies
**Hypothesis: the high number of primers/template strand on beads outcompetes the acrydite primers in gel
*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)
*Dr. Zhang suggested adding 1/10th (50nM) of free floating Acrydite primer to help efficiency
**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
 
===Harvard Protocol===
#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-->

Latest revision as of 21:19, 15 June 2017

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. 
  1. Let gel slide dry in AirClean hood for 30min
  2. 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)
  3. Pipet 25ul onto center of gel
  4. Apply 18x30mm cover slip
  5. Apply an orange SecureSeal chamber
  6. Fill chamber with mineral oil and seal holes with stickies.
  7. Slide PCR on Biorad thermocycler
 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
  1. 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