Matt:LabNotes/2017-6-1: Difference between revisions
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>Mzcai (Created page with "=Image and Seq= 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== *Gels w...") |
>Mzcai m (→Gel PCR) |
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#Slide PCR on Biorad thermocycler | #Slide PCR on Biorad thermocycler | ||
94C 3min -> (94C | 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold | ||
#Place directly on Olympus with GFP/FITC filter | |||
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#Remove SecureSeal and put slide in glass Coplin jar of Hexane for 10min (should be 5min) | #Remove SecureSeal and put slide in glass Coplin jar of Hexane for 10min (should be 5min) |
Revision as of 23:03, 2 June 2017
Image and Seq
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
- Gels were cast Matt:LabNotes/2017-5-24 and stored in water
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 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
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 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
- Place directly on Olympus with GFP/FITC filter