Matt:LabNotes/2017-6-14
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Image and Seq[edit]
- Last time
- messed up the sample with USER in PCR mix
- pre-nicking with USER did not work
http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm
Sample Conditions[edit]
3 Samples:
- Include 50nM Acrydite primer
- Include USER enzyme in the PCR mix
- USER will nick the biotinylated strand leaving only: CAGTGT
- Include 0.1% Tween-20 and 0.2% BSA
- These are included in the Harvard protocol
Cast 10% gels with primer[edit]
- 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[edit]
- Beads prepared on Matt:LabNotes/2017-5-24
Gel PCR[edit]
- Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
- Prepare 25ul PCR mix Sample1
- 2.5ul 5uM Primer2
- 1.25ul 1uM Acrydite Primer (50nM final)
- 12.5ul KAPA SYBR FAST MM
- 5ul beads (1:1 mix of v1 and v3)
- 3.75ul H2O
- Prepare 25ul PCR mix Sample2
- 2.5ul 5uM Primer2
- 12.5ul KAPA SYBR FAST MM
- 1.25ul USER
- 5ul beads (1:1 mix of v1 and v3)
- 3.75ul H2O
- Prepare 25ul PCR mix Sample3
- 2.5ul 5uM Primer2
- 12.5ul KAPA SYBR FAST MM
- 5ul beads (1:1 mix of v1 and v3)
- 2.5ul 1% Tween-20
- 2.5ul 2% BSA
- 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 2 first, put in sample 1 & 3 after 37C 30min incubation
37C 30min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
- Place directly on Olympus with GFP/FITC filter
Results[edit]
- All 3 had deformed SecureSeals and were leaking oil
- Used 20X objective to image
Sample 1[edit]
File:20170614 Sample1 2ndprimer composite.jpg
Sample 2[edit]
- Spots about 4um in diameter
File:20170614 Sample2 USER composite.jpgFile:20170614 Sample2 USER1 composite.jpg
Sample 3[edit]
File:20170614 Sample3 Tween20 composite.jpg
Conclusion[edit]
- Sample 2 with USER looks promising but could also just be signal similar to 1 cycle
- Sample 1 and 3 are not polonies
- Did some research into acryloyl groups and acrydite-modified primers. Very unstable and should not be stored at 4C like I have been. Order new acrydite-primers and aliquot, store at -20C
- Tm of Primer2 is 53C... so try decreasing annealing temp of PCR to 45C