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- Digested ‘A&B’ and ‘C’
- Trying a different method for attaching ‘A&B’ w/ C
- ‘A&B’ cut w/ Spe & Pst
- ‘C’ cut w/ Xba & Pst (insert)
- When these parts were ligated together, there were many colonies on the experimental plate, few colonies on the vector control, but many colonies on the insert colonies.
- We had a theory that since Part C and Part C plasmid are very close in bp, we may have extracted both pieces when gel extracting.
- We wanted to do a PCR screen, but we ran into a problem, seeing as there were no bands – at all. Even if there is no product, you would see primers, but the primers here were very faint. Likewise, PCR screening was not working when screened with other parts as well.
- Time for an investigation:
- Run test on PCR screening process
- 1. Old forward 1/10 dilution primer 1 uL
- 2. Old reverse 1/10 dilution primer 1 uL
- 3. New forward 1/10 dilution primer 1 uL
- 4. Old reverse 1/10 dilution primer 1 uL
- 5. 100% oligos of forward primer 1 uL
- 6. 100% oligos of reverse primer 1 uL
- 7. 1/10 dilution of forward (1 uL), 1/10 dilution of reverse (1 uL) in 18 uL dH2O (old primers)
- 8. 1/10 dilution of forward (1 uL), 1/10 dilution of reverse (1 uL) in 18 uL dH2O (new primers)
- 9. Re-PCR’ed parts from yesterday that failed 5 uL
- a. Maybe it wasn’t run long enough because Taq was compromised
- b. Yesterday rano n 25-27 cycles, today ran extra 18 cycles
- 10. Run on a 2% agarose gel w/ 100 bp & 2 log ladder
- Results: oligos may not be good.
- Run test using Taq enzymes
- Tubes 1-8 of PCR screen performed on 8/18/10 were run with 4:30 extension time at ~27 cycles. Today taken out and placed back into PCR machine. Ran another PCR screen program with 4:30 extension time for another 18 cycles. Then ran on a 2% agarose gel with 100bp ladder and 2 log ladder.
- Results: order new Taq?
- Conclusion: no conclusion can be drawn. The test gives very contradictory information.
- Meanwhile, I746352-B0034, E1010-PSP, C0051-PSP, B0034-C0052 were ligated in attempts to investigate the leaky expression phenomena.
- Initiator/las receiver progress: Successfully ligated R0082 with RBS-C0051-B0015 and I14015 with RBS-C0051-B0015. We may have two complete parts because there were practically no colonies on the control plates. Of course, it also depends on how our sequencing results go because we can’t PCR screen this until we figure out what’s wrong.
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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