Matt:LabNotes/2017-6-27
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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]
- 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 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)
- 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
Cast 10% gels with primer[edit]
- 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
- Add 18ul to oval and cover with coverslip
- Put slides in argon chamber and polymerize 30min
- Shake in H2O 30min
Gel PCR[edit]
- Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
- 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
- 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.
- Place one slide on magnet for 1min
- Slide PCR on Biorad thermocycler
37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
- 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
- Just GFP: File:Magnet Pos2 GFP.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