Team:Columbia-Cooper/GenspaceNotebook
Genspace Notebook
July 5
- Grew out BBa_k325219 and BBa_k325909 in media C.
- Restriction digest of BBa_k231000 and BBa_k231001 with EcoRI and PstI
July 15
- CDS7 isolation and ligation
July 19
- NcoI and BamHI digest of pET Vector
July 20
- Another attempt at NcoI and BamHI digest of pET Vector
July 21
- Gel Purified pET Vector Digest
- Grew out more pET vector
July 24
- Received new NcoI and BamHI enzymes
- pET Vector digest with new NcoI and new BamHI
- pET Vector digest with old NcoI and old BamHI
- pET Vector digest with new NcoI and another with old NcoI
- pET Vector digest with BamHI and anther with old BamHI
July 25
- Ran a gel with all pET Vector digests from July 24th
- Fermentas transformation on pET Vector
July 28
- CDS7 digest with NcoI and BamHI
- Prepared plates of kanamycin and pET Vector
- Oligo annealed with Silver Lab protocol
- Maxiprep using ~50mL CDS7 growth
- pET Vector inoculation
July 29
- pET Vector digest with BamHI and NcoI
- Gel purification of pET Vector digest
- Inoculated more pET Vector
- Inoculated C-media for transformations
- Miniprep of pET Vector
- Ligated 4 oligos
- Prepare transformation ready cells using C-media culture prepared the previous day
July 30
- Miniprep of pET Vector
- Digestion of pET Vector with NcoI and BamHI
- Oligo PCR
-Digest pUC19 with EcoRI and PstI
August 1
- Ran gels to verify pET and PUC double digests
- Restriction digest of annealed oligo sequences with EcoRI and PstI
- Oligo PCR verification
- Digested pSB1C3 with EcoRI and PstI
August 2
- pUC19 and pET Vector were isolated and gel purified
- Miniprep of pUC19, pET Vector, and pSB1C3
August 3
- Ligated pUC19 and all the oligos
August 5
- PCR with new CDS7 was successful. Sung has “never seen a brighter 70 bp lane in [his] life”
- pUC19 – quantum dot synthesis oligos worked
- Miniprep, digest with EcoRI and PstI, and gel run of CDS7
August 6
- Gel extraction of pUC19 and pET Vector compatible CDS7 amplicon
August 7
- Inoculated oligo ligation cells in ampicillin media
- Digested and ran more of oligo ligations
- Inoculated more of the ligations
- Inoculated 6 CDS7 colonies
August 8
- Optimized PCR
August 22
- Oligo annealed
- Ethanol precipitation of DNA
August 23
- Precipitation of CDS7 and restriction digest with EcoRI and PstI
- Oligo annealed
August 24
- Dephosphorylated backbone and created ligation mixtures
- Annealed biobrick part
- Made master mix for biobrick backbone
- Oligo annealed
- Digested pUC19 with EcoR1 and PstI
August 30
- Attempted ligation of biobrick
August 31
- Tested previous day’s ligation in a gel and it failed
September 1
- pUC19 digest with EcoRI and PstI
- Gel purification of pUC19 digest
- Alcohol precipitated CDS7
September 2
- Received new primers
- Ran three PCR reactions, CDS7 forward with RBS, reverse, and control
- Ran 1/100 dilution of gel
September 7
- Another CDS7 PCR
September 8
- Restriction digest of J140 with EcoRI and PstI
September 9
- Ran gel of pET Vector, j140, pSB1C3, CDS7, and CDS7S
September 13
- Restriction digest of master mix with EcoR1 and Pst1
- Made a ligation mix
- Transformed with ligation mix
September 14
- pET Vector digest with Nco1 and BamH1
- CDS7 PCR
- CDS7 digest with EcoRI and PstI
- CDS7 digest with XbaI and PstI
- Gel verification and purification of pET Vector and CDS7
- Ligation of CDS7 EcoRI and PstI cut with pSB1C3
September 20
- VF and VR PCR verification
- Restriction digest on pSB1C3 with Pst1 and EcoR1
- Gel verification of digest
September 21
- Verified base pair numbers of primers and verified they work
- PCR of CDS7
- Gel purification of CDS7
- Digest CDS7 with EcoR1 and Pst1
- Phosphotased pSB1C3
- Made ligation mix with all the parts
- Transformed with the ligation mix
September 23
- Ligation mix from previous day failed
- Re-did everything from September 21st except this time made a new master mix with different PCR settings
- Ligated and transformed with the new master mix
September 24
- Grew colonies overnight from previous days ligation and transformation
- Grew out colonies with LB chloramphenicol
- Made minipreps of CDS7
September 26
- PCR a sequencing segment from plasmid with VF2 and VR
- Sent segment off for sequencing
- Reviewed other transformations