Matt:LabNotes/2017-6-27: Difference between revisions

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http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm
http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm


*Use new acrylamide primer
*[[Matt:LabNotes/2017-6-14|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
*Annealing temp of PCR changed from 55C -> 45C
*One sample placed on magnet for 1min
*One sample placed on magnet for 1min
*One sample no magnet
*One sample no magnet
*K


==Prepare Beads==
==Prepare Beads==
Line 39: Line 41:


==Gel PCR==
==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 within 5 minutes of liquid film disappearance
 
#Let gel slide dry in AirClean hood for 30min
#Prepare 25ul PCR mix per gel
#Prepare 25ul PCR mix per gel
#*2.5ul 5uM Primer2
#*2.5ul 5uM Primer2
#*12.5ul KAPA SYBR FAST MM
#*12.5ul KAPA SYBR FAST MM
#*10ul beads (1:1 mix of v1 and v3)
#*5ul beads (1:1 mix of v1 and v3)
#*1.25ul USER
#*3.75ul H2O
 
#Pipet 25ul onto center of gel
#Pipet 25ul onto center of gel
#Apply 18x30mm cover slip
#Apply 18x30mm cover slip

Revision as of 23:03, 28 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

  • 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

  1. 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
  2. Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
  3. Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
  4. Vortex
  5. Pull down 1min on magnet
  6. Remove supernatant
  7. 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)
  8. Add to beads, pipette mix, and incubate 15min at room temp
  9. Pull down
  10. Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
  11. Bring volume to 40ul with H2O

Cast 10% gels with primer

  1. 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
  2. Add 18ul to oval and cover with coverslip
  3. Put slides in argon chamber and polymerize 30min
    • Shake in H2O 30min

Gel PCR

  1. Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
  2. 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
  1. Pipet 25ul onto center of gel
  2. Apply 18x30mm cover slip
  3. Apply an orange SecureSeal chamber
  4. Fill chamber with mineral oil and seal holes with stickies.
  5. Place one slide on magnet for 1min
  6. Slide PCR on Biorad thermocycler
 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
  1. Place directly on Olympus with GFP/FITC filter