Matt:LabNotes/2017-5-24
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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
Cast 10% gels with primer[edit]
- Make Gel Mix
- 25ul 199:1 40% A:B mix
- 2ul 10% BSA
- 67ul H2O
- 2ul 50uM Acrydite primer
- 2ul 5% TEMED
- 2ul 5% APS
- Add 18ul to oval and cover with coverslip
- Put slides in argon chamber and polymerize 30min
- Put in MilliQ H2O overnight
- Normally Wash in H2O 30min shaker
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 45sec -> 55C 30sec -> 72C 1min) x 7 -> 72C 3min -> 4C hold
- Remove SecureSeal and put slide in glass Coplin jar of Hexane for 10min (should be 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 Coplin jar of Wash 1E twice for 4min
- Store in Wash 1E
Strip Second Strand[edit]
At the conclusion of the PCR reaction, amplicons are present in double-stranded form, with one strand anchored to the gel via the acrydite modification. Denaturation will permit us to remove the un-anchored strand, such that the remaining strand can be free to serve as a template for hybridization and sequencing reactions.
- Apply blue FrameSeal chamber
- Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
- Wash twice with 30% formamide in 2X SSC
- Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
- Wash twice with 30% formamide in 2X SSC
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
FISH[edit]
- Prepare 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC
- Add probes preheated to 75C and incubate 10min at room temp
- Wash with 2X SSC twice
- Add 2X SSC and then cover with coverslip
- Image
Results[edit]
- Didn't see anything under confocal in either channel
- When trying to remove coverslip to stain with SYBR gold the sample got ruined
Next Time[edit]
- Try imaging directly after PCR (using SYBR Green as dye)
- That way can keep increasing number of cycles