Revision as of 00:34, 29 September 2011 by Mjp2175 (Talk | contribs)

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 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