iGEM 2016 — Team TP_CC_SanDiego

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# Team:TP CC SanDiego/Notebook

###### NOTEBOOK

T P - C C

[HOME](https://2016.igem.org/Team%3ATP_CC_SanDiego)
[PROJECT](https://2016.igem.org/Team%3ATP_CC_SanDiego/Description)
[NOTEBOOK](https://2016.igem.org/Team%3ATP_CC_SanDiego/Notebook)
[OUTREACH](https://2016.igem.org/Team%3ATP_CC_SanDiego/Practices)
[TEAM](https://2016.igem.org/Team%3ATP_CC_SanDiego/Team)
[ATTRIBUTIONS](https://2016.igem.org/Team%3ATP_CC_SanDiego/Attributions)
[COLLABORATIONS](https://2016.igem.org/Team%3ATP_CC_SanDiego/Collaborations)

[BACK TO TOP](#top)
[DAY 3](#week3)
[DAY 6](#week6)
[DAY 9](#week9)
[DAY 12](#week12)
[DAY 15](#week15)

[Click Here for the full notebook PDF.](https://static.igem.org/mediawiki/2015/7/72/TP_CC-SanDiego_Lab_Notebook.pdf)

##### DAY 1

SUMMARY (July 15th):

* PCR Amplified pPBR322-based plasmid backbone, pBAD from E. coli gDNA, and rrnB from the F plasmid.
* DPN1 digest for 1 hour at 37°C and PCR Purification using Qiagen Spin Columns.
* Gel analysis for correct PCR amplification of fragments

WHAT TO DO NEXT:

* Gibson Assembly all fragments into complete plasmid for diagnostics.

##### DAY 2

SUMMARY (July 18th):

* Gibson Assembly of vector backbone, pBAD, rrnB, and placeholder YFP (in between pBAD and rrnB) gene fragments (30 fmol each).
* Electroporated assemblies into electrocompetent Epi300 cells; recovered for 1 hour at 37°C and plate on LB+Tet plates.

WHAT TO DO NEXT:

* Colony PCR of colonies on plate.

##### DAY 3

WHAT WE FOUND:

* Numerous colonies (~40) to screen.

SUMMARY (July 19th):

* Colony PCR of 16 colonies using screening primers to test accurate assembly across pBAD-YFP-rrnB junctions.

WHAT TO DO NEXT:

* Restart positive colonies for miniprep.

##### DAY 4

SUMMARY (July 20th):

* Restarted four positive colonies in 5 mL of LB+Tet in 37°C for minipreps tomorrow by sampling from patches made during colony PCR.

WHAT TO DO NEXT:

* Plasmid miniprep from restarted cultures.

##### DAY 5

SUMMARY (JULY 21st):

* Extracted pBAD plasmids from cultures using Qiagen Miniprep kit; plasmid extracts were nanodropped to find plasmid concentrations.
* Plasmids were HindIII digested to ensure final proper plasmid construction.

WHAT TO DO NEXT:

* Amplify vector backbone for chitinase plasmids; replace YFP with chitinase inserts.

##### DAY 6

SUMMARY (AUGUST 12th):

* Designed inserts were ordered from IDT (LbCHI31 and LbCHI32)

* red pcr tube in -20C on igem rack contains product

WHAT TO DO NEXT:

* Transformation into BL21 and Dh5Alpha competent cells
* Make gels

##### DAY 7

SUMMARY (August 15th)

* Inserts arrived from IDT; resuspended in H2O.
* Gibson Assembly of LbCHI31 and LbCHI32 fragments into pBAD vector backbone.
* Electroporated assemblies in electrocompetent Epi300 cells and recovered for 1 hour at 37°C before plating on LB+Tet plates.

WHAT TO DO NEXT:

* Colony PCR of LbCHI31 and LbCHI32 plasmid transformed colonies.

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