IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 5

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Contents

7/16/07

Colony PCR of R-P constructs

Stephanie Colony PCR'ed the following:

Stephanie's notebook entry

Plate Reader (Light Scattering)

Stephanie set up another plate reader for fluorescence. One of the things she is testing is choosing ridiculous wavelengths to see if we can get light scattering data that correlates to cell growth.

Stephanie's notebook entry Results posted by Stephanie

Miniprep of the J04450 and JT constructs

In preparation for building the constitutive RFP + JT construct, George miniprepped the following parts which were grown overnight:

George's notebook entry

Digesting the J04450 and J-T

George digested the J04450 with SpeI and PstI; he also digested the J23039>T9002 with XbaI and PstI.

George's notebook entry

Sequencing the Promoters: R0011, R0051, R0052

George and Stephanie sent the R0011, R0051, and the R0052 for sequencing.

Dephosphorylating the J04450 and J-T

George dephosphorylated the J04450 and the J-T with Antarctic Phosphotase and then heat inactivated the AP. Silly George, AP is for vectors not inserts. Redo it tomorrow.

George's notebook entry

Liquid Cultures of R-P constructs and J-T

George grew liquid cultures of the following:

The Upper/Lower Right is due to the fact that the colony PCR plate had duplicates of those colonies and it was unclear which colony was correct (at least, the PCR and gel was correct). So, George took both samples and labeled one as coming from the upper right of the plate (when viewed with the handwriting going the correct way) versus lower right.

George's notebook entry

7/17/07

Miniprepping the R-P and JT

George miniprepped the R-P and J23039 <T9002 constructs that were grown overnight in preparation for building the RFP+JT and tetR+JT constructs.

George's notebook entry

Digesting the R-P and JT

George digested the R-P constructs with SpeI and PstI; he also digested the J23039 <T9002 with XbaI and PstI in preparation for building the RFP+JT and tetR+JT constructs.

George's notebook entry

Sequencing the R-P constructs

George sent off sequences for the R-P constructs

George's notebook entry

Dephosphorylating the R-P constructs

George dephosphorylated the R-P constructs with Antarctic Phosphotase and then he heat inactivated the AP.

George's notebook entry

Clonewell and Vacufuge of R-P constructs and JT

George clonewelled and vacufuged the R-P constructs. Unfortunately, he forgot to include JT and had to re-use the same gel.

George's notebook entry

Ligation and Transformation tetR-JT and RFP-JT constructs

George Roche ligated the following:

and then transformed them into BL21.

User:GeorgeXu#Ligation_and_Transformation_tetR-JT_and_RFP-JT_constructs

7/18/07

Colony PCR of tetR-JT and RFP-JT

George colony PCR'ed the tetR-JT and RFP-JT constructs.

7/20/07

Liquid Cultures of R-P and JT parts

George grew liquid cultures of all the working R-P parts (according to the colony PCR). He discovered that the R52's were incorrect (sequences + wrong selection plate). The parts he grew up were:

7/21/07

Miniprep of R-P and JT

Stephanie miniprepped the parts that were grown in liquid culture.

Stephanie's notebook entry

Digestion of R-P and JT

Stephanie digested the parts that were miniprepped.

Stephanie's notebook entry

Dephosphorylation of R-P and JT

George dephosphorylated the JT with Antarctic Phosphotase.

George's notebook entry

Clonewell and Vacufuge of R-P and JT

George clonewelled and vacufuged the R-P and JT

George's notebook entry

7/22/07

Colony PCR of RP-JT parts

George colony PCR'ed the RP-JT parts for the third time. Unfortunately, the results were horrible.

|George's notebook entry

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