Matt:LabNotes/2017-6-13
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Image and Seq[edit]
- Last time 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[edit]
- 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[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
For USER prenicked Beads (Sample 2)[edit]
- Take 5ul beads (1:1 mix of v1 and v3)
- Add 2.5ul 10X Cutsmart Buffer
- Add 2.5ul USER
- Add 15ul H2O
- Incubate 15min @ 37C
- Put on cold block 5min
- Pull down beads, remove supernatant, and resuspend in 5ul Dynabeads buffer
- Was not able to pull down beads so just pipetted the reaction mix (1/5 diluted beads)
Gel PCR[edit]
- 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)
- 5ul H2O
- For USER sample 3 include 1.25ul USER and use 3.75ul H2O 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 15min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
- Made a mistake and did 15sec 37C at first, didn't catch the mistake until after 94C 3min incubation probably killing the USER enzyme
- Restarted the thermalcycle program anyway just in case but probably won't work
- Place directly on Olympus with GFP/FITC filter
Results[edit]
Sample 1: Standard[edit]
- Like seen before, amplification on beads only
File:20160613 Sample1 Standard composite.jpg
Samples 2: Prenick with USER[edit]
- Signal only in beads
File:20160613 Sample2 PreUSER2 composite.jpgFile:20160613 Sample2 PreUSER5 composite.jpg
Sample 3: USER in PCR[edit]
- Again signal only in beads
- Expected because not enough time for USER digest
File:20160613 Sample3 USER composite.jpgFile:20160613 Sample3 USER1 composite.jpg450px
Conclusion[edit]
- Need to try USER in PCR again
- Try adding 1/10th free floating acrydite primer
- Try following Harvard protocol more closely
- Fresh gels made no difference