Matt:LabNotes/2017-5-24
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http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm
Cast 5% gels with primer
25ul ABD mix 2ul 10% BSA 67ul H2O 2ul 50uM 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
- 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.
Prepare 25ul PCR mix per gel
- 2.5ul 5uM Primer2
- 12.5ul KAPA SYBR FAST MM
- 10ul beads
- Pipet 25ul onto center of gel
- Apply 18x30mm cover slip
- Apply an orange SecureSeal chamber
- Fill chamber with mineral oil. This is a little tricky but you get good with practice. Use a P1000 pipetman filled with ~550 uL of mineral oil. Use a Chemwipe to dry off the tip of the pipet tip. The goal is to get the mineral oil in without spilling any near the surface of the hole (as this makes it more of a pain when you go to seal the hole with the stickies). A good technique to avoid spilling is to hold the slide tilted at a 45-degree angle while filling. Use the lower hole (in whatever direction you are holding the slide) to fill. It is OK to leave some air near the upper hole- more important to avoid letting oil seep out of it. When sufficient oil is in, turn the slide flat (0 degrees) before withdrawing the pipetman from the hole.
- Wipe off any excess mineral oil from the surface of the SecureSeal, if neccessary. Seal holes with stickies.
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 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