Matt:LabNotes/2017-6-27
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http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm
http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm
- Use new acrylamide primer
- Annealing temp of PCR changed from 55C -> 45C
- One sample placed on magnet for 1min
- One sample no magnet
Prepare Beads
- 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
- 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
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.
- Place one slide on magnet for 1min
- Slide PCR on Biorad thermocycler
94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
- Place directly on Olympus with GFP/FITC filter