Team:Penn/Notebook
From 2013.igem.org
- 4-Jun
- Learned how to make competent cells, growing up two strains for tomorrow
- Transformed 8 plasmids
- Determined EL222 fusion is risky but still going ahead with it
- Linkers are totally setlled
- Found zinc finger plasmid and updated target sequence
- Learned how to make tetr- mcherry fusion
- Settled on 5 promoters
- 5-Jun
- Learned how to make competent cells, testing them and then making more tomorrow
- Transformed 8 plasmids again
- Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system, ready to order
- Made ultramers for variable promoter blocks (and no target neg controls) – ready to order
- Spilled a lot of iced tea outside, bummer
- Started primers for dna binding machines
- Got a handle on cas9 fusions (pun intended).
- Put awesome pics in dropbox
- 6-Jun
- Clean up dropbox
- Update budget sheet with addgene and cell center orders
- Finish primers for fusion
- Set up plate reader for GFP and mCherry assays
- run minipreps on pdawn, pdawn-mcherry, pet26b
- Grow up mCherry stock
- Wrote Penn iGEM on our plasmid
- 7-Jun
- Transform
- C0012 –amp/chlor (do both)
- M11307 – amp/chlor (do both)
- I13458 – amp/chlor (do both)
- R0010 – amp/chlor (do both)
- R0051 – amp
- K206000 –chlor
- Start the LIMS and file all the strains and DNA we have made/ ordered
- Mini-preped
- I9002
- I13458
- C0051
- Pdawn-mcherry
- Pdawn
- Pet26b
- Dhsa mcherry
- Pdawn dhsa
- Psb1a3
- JM mcherry
- 8-Jun
- Miniprep Addgene stuff + transformations that worked
- Growing up low copy plasmids in 40mLs
- 8-Jun
- Miniprep Addgene stuff + transformations that worked
- Growing up low copy plasmids in 40mLs
- Transformed everything that has failed
- 17-Jun
- Grow up luxI culture and grow up tetR culture
- Sequence all of the minipreps
- Transform t9002 in psb1A3 in NEB10
- Retransform ptetGFP to see if BL21DE3 cells are competent
- Transform r0079, k081015, r0063 in NEB10
- Miniprep psb1k3
- Redo dam gel with more dna
- Figure out second control zfp from addgene
- Figure out how to add luxR binding site to target region
- Order sequencing primers for all addgene minipreps
- Bisulfite converted msssi methylated c0051
- 18-Jun
- The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results
- Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit
- Order 13420 (second zfp)
- Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)
- 19-Jun
- Transform failed transformation
- Make competent DH5a && Dam-
- Figure out methylation assays for promoters
- Miniprep psb1A3 && all the 40mL cultures
- Picked many colonies
- Check pTet-gfp under blue light
- 20-Jun
- run plux-luxI pcr
- run pdawn-luxI pcr
- run pDawn-tetR pcr
- run pet26b-tetR pcr and
- run pDawn-GFP pcr
- run pDawn-mCherry-secretion tag pcr
- Nano drop last nightÕs mini preps to check for accuracy
- Culture amp resistant successful transformations
- Make 5 L LB
- Miniprep all the successful transformations w/ new protocol
- 21-Jun
- Jun troubleshoot plux-luxI pcr
- roubleshoot pdawn-luxI pcr
- made pDawn-tetR pcr work
- troubleshoot pet26b-tetR pcr
- troubleshoot pDawn-GFP pcr
- troubleshoot pDawn-mCherry-secretion tag pcr
- miniprep growing cultures, be sure to pick only the glowing ligations
- ransform the correct t9002 amp ligation - determined from gel
- digested t9002 in amp and ptet gfp in amp to identify the correct ligation
- all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest
- 24-Jun
- Dam-/dh5a v dam methylation + dpnI && dpnII digest
- Digested/ligated/transformed t9002 in amp
- Get methylated biobrick sequenced
- get chlor backbones sequenced
- culture t9002 transformations in liquid media with i751250
- mini prep stuff in the incubator
- figure out the primer issues 8
- Pick t9002 colonies for miniprep
- 26-Jun
- Dam-/dh5a v dam methylation + dpnI && dpnII digest
- Digested/ligated/transformed t9002 in amp
- get chlor backbones sequenced
- culture t9002 transformations in liquid media with i751250
- mini prep stuff in the incubator
- figure out the primer issues
- Pick t9002 colonies for miniprep
- USER Cloning reporter plasmid