Matt:LabNotes/2017-6-27: Difference between revisions

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>Mzcai
(Created page with "=Image and Seq= http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm <br> http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm *Use new acrylamide primer *Anne...")
 
>Mzcai
 
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http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm
http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm


*Use new acrylamide primer
*[[Matt:LabNotes/2017-6-14|Last time]]
*Use new acrylamide primer (needs to be stored in -20C, aliquoted to avoid freeze/thaw)
*Annealing temp of PCR changed from 55C -> 45C
*Annealing temp of PCR changed from 55C -> 45C
*One sample placed on magnet for 1min
*One sample placed on magnet for 1min
*One sample no magnet
*One sample no magnet
*K


==Prepare Beads==
==Prepare Beads==
Line 39: Line 41:


==Gel PCR==
==Gel PCR==
  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 within 5 minutes of liquid film disappearance
 
#Let gel slide dry in AirClean hood for 30min
#Prepare 25ul PCR mix per gel
#Prepare 25ul PCR mix per gel
#*2.5ul 5uM Primer2
#*2.5ul 5uM Primer2
#*12.5ul KAPA SYBR FAST MM
#*12.5ul KAPA SYBR FAST MM
#*10ul beads (1:1 mix of v1 and v3)
#*5ul beads (1:1 mix of v1 and v3)
#*1.25ul USER
#*3.75ul H2O
 
#Pipet 25ul onto center of gel
#Pipet 25ul onto center of gel
#Apply 18x30mm cover slip
#Apply 18x30mm cover slip
Line 53: Line 56:
#Slide PCR on Biorad thermocycler
#Slide PCR on Biorad thermocycler
    
    
   94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
   37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 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==
==Results==
#Remove SecureSeal and put slide in glass Coplin jar of Hexane for 5min
===Control (no magnet)===
#Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
[[File:Composite_Control_Pos1.jpg|450px]][[File:Composite_Control_Pos2.jpg|450px]]
#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==
===Magnet===
#Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
[[File:Composite_Magnet_Pos1.jpg|450px]][[File:Composite_Magnet_Pos2.jpg|450px]]
#Wash twice with Wash 1E


==Hybridize FISH probes==
*Just GFP: [[File:Magnet_Pos2_GFP.jpg|450px]]
#Add 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC preheated to 80C and incubate 10min at room temp
===Special Note===
#Wash twice with 2X SSC
*The z plane with fluorescence in focus is not the same as when beads are in focus
#Add 2X SSC and then seal with glass coverslip
*In these images, the in focus BF bead images was merged with in focus fluorescent images
 
*What the BF channel looks like when the GFP channel is in focus:
==Results==
[[File:Composite_Magnet_Pos3.jpg|450px]]
===SYBR Green===
[[File:20170604_w2_composite.jpg|650px]]<br>
[[File:20170605_w2_composite.jpg|650px]]<br>
[[File:20170606_w2_composite.jpg|650px]]<br>
===FISH===
[[File:20170607_w1_composite.jpg|650px]]
*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==
==Conclusion==
*Initially thought the SYBR Green images looked good
*Probably not rolonies, all fluorescent signal has similar characteristics that I have seen in previous tries:
**The fluorescence signal was centered around beads and had diameter of ~8um after 15 cycles
**Bright star shapes that look kind of like cell membrane stretched over cytoskeleton
**Only a fraction of beads had fluorescence suggesting it was not autofluorescence and true polonies
**Sometimes the star is surrounded by a fainter round signal
*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


===Harvard Protocol===
*Do No Bead Control to see if crap is still here
#Prepare 70% formamide 1X SSC in plastic coplin jar
*Vary bead concentration
#*4mL 20X SSC
*Dr. Zhang says 50-100um polonies is very normal size
#*56mL formamide
**Smaller template has larger polonies because it can diffuse easier
#*20mL H2O
*Do 50+ cycles overnight
#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 02:17, 12 July 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

  • Last time
  • Use new acrylamide primer (needs to be stored in -20C, aliquoted to avoid freeze/thaw)
  • Annealing temp of PCR changed from 55C -> 45C
  • One sample placed on magnet for 1min
  • One sample no magnet
  • K

Prepare Beads[edit]

  1. 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
  2. Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
  3. Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
  4. Vortex
  5. Pull down 1min on magnet
  6. Remove supernatant
  7. Combined 15ul buffer with 15ul amplicon
    • BarcUv1 500nM (dsDNA PCR product with Uracil prepared by Dan 5.25.2017)
    • BarcUv3 500nM (dsDNA PCR product with Uracil prepared by Dan 5.25.2017)
  8. Add to beads, pipette mix, and incubate 15min at room temp
  9. Pull down
  10. Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
  11. Bring volume to 40ul with H2O

Cast 10% gels with primer[edit]

  1. Make Gel Mix
    • 12.5ul 199:1 40% A:B mix
    • 1ul 10% BSA
    • 33.5ul H2O
    • 1ul 50uM Acrydite primer
      • Stored in -20C
    • 1ul 5% TEMED
    • 1ul 5% APS
  2. Add 18ul to oval and cover with coverslip
  3. Put slides in argon chamber and polymerize 30min
    • Shake in H2O 30min

Gel PCR[edit]

  1. Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
  2. Prepare 25ul PCR mix per gel
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 5ul beads (1:1 mix of v1 and v3)
    • 1.25ul USER
    • 3.75ul H2O
  1. Pipet 25ul onto center of gel
  2. Apply 18x30mm cover slip
  3. Apply an orange SecureSeal chamber
  4. Fill chamber with mineral oil and seal holes with stickies.
  5. Place one slide on magnet for 1min
  6. Slide PCR on Biorad thermocycler
 37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
  1. Place directly on Olympus with GFP/FITC filter

Results[edit]

Control (no magnet)[edit]

File:Composite Control Pos1.jpgFile:Composite Control Pos2.jpg

Magnet[edit]

File:Composite Magnet Pos1.jpgFile:Composite Magnet Pos2.jpg

Special Note[edit]

  • The z plane with fluorescence in focus is not the same as when beads are in focus
  • In these images, the in focus BF bead images was merged with in focus fluorescent images
  • What the BF channel looks like when the GFP channel is in focus:

File:Composite Magnet Pos3.jpg

Conclusion[edit]

  • Probably not rolonies, all fluorescent signal has similar characteristics that I have seen in previous tries:
    • Bright star shapes that look kind of like cell membrane stretched over cytoskeleton
    • Sometimes the star is surrounded by a fainter round signal
  • Do No Bead Control to see if crap is still here
  • Vary bead concentration
  • Dr. Zhang says 50-100um polonies is very normal size
    • Smaller template has larger polonies because it can diffuse easier
  • Do 50+ cycles overnight