Matt:LabNotes/2017-6-27: Difference between revisions
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>Mzcai (Created page with "=Image and Seq= http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm <br> http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm *Use new acrylamide primer *Anne...") |
>Mzcai mNo edit summary |
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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== | ||
#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 | ||
#* | #*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
- 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
- Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
- 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
- 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