Matt:LabNotes/2017-6-13
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- Matt:LabNotes/2017-6-8 confirmed no polonies
- Ideas for why:
- Gels were cast and stored poorly in water (UV slides beforehand, use immediately or store in buffer with EDTA)
- Primers on beads outcompeted primers in gel (USER digest amplicons on beads to shorten primer on beads)
- Ideas for why:
http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm
Sample Conditions
- 3 samples to test how USER can help create polonies
- USER will nick the biotinylated strand leaving only: CAGTGT
Samples:
- Basic repeat of last time with fresh gels
- USER digest beads before adding to gel
- Include USER enzyme in the PCR mix
Cast 10% gels with primer
- Make Gel Mix
- 25ul 199:1 40% A:B mix
- 2ul 10% BSA
- 67ul H2O
- 2ul 50uM Acrydite primer
- 2ul 5% TEMED
- 2ul 5% APS
- Add 18ul to oval and cover with coverslip
- Put slides in argon chamber and polymerize 30min
- Wash in H2O 30min shaker
Prepare Beads
- Beads prepared on Matt:LabNotes/2017-5-24
For USER prenicked Beads (Sample 2)
- Take 10ul beads (1:1 mix of v1 and v3)
- Add 5ul 10X Cutsmart Buffer
- Add 5ul USER
- Add 30ul H2O
- Incubate 15min @ 37C
- Put on cold block 5min
- Pull down beads, remove supernatant, and resuspend in 10ul Dynabeads buffer
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
- 10ul beads (1:1 mix of v1 and v3)
- For USER sample 3 include 1.25ul USER and use 1.25ul 10uM Primer2 instead
- 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
- Put in sample 3 first, put in sample 1 & 2 after 37C 30min incubation
37C 30min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
- Place directly on Olympus with GFP/FITC filter