Team:CSU Fort Collins/Notebook/HVP/Jun

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Revision as of 18:06, 17 October 2014

High-Value Product Notes - June

High-Value Product Daily Notes

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Jun

pMBI Assembly

July

pMBI and pMevT Assembly

August

Troubleshoot Everything!

September

Plan B

JUNE

Monday, June 2 - Friday, June 6

Researched potential products and decided on the addition of the mevalonate pathway in order to direct E. coli to terpenoid production.

Monday, June 9 - Monday, June 16

Researched the mevalonate pathway in E. coli and sequenced the genes to be inserted into puc19. Designed primers as specified in the Gibson Assembly Protocol for transformation of the mevalonate pathway from yeast into E. coli.

Thursday, June 19

Ran a gel electrophoresis to try and confirm presence of DNA in obtained yeast samples. No results.

Tuesday, June 24

Mixed and autoclaved two 500 mL bottles of LB Broth with Agar and ampicillin. Poured two sleeves of plates to use for culturing.

Wednesday, June 25

Took a small sample of E. coli with puc19 from a previous colony and suspended in LB Broth. Shook at 225 rpm in incubator at 37° C overnight to culture.

Thursday, June 26

Completed a miniprep of E. coli with puc19 from the overnight culture. Followed plasmid miniprep protocol as given in the Informatics (Life Technologies) kit.

Friday, June 27

Completed PCR with the 5 pieces according to the mixture of New England Biotechnologies' Q5 2X Master Mix protocol. Ran a gel electrophoresis to confirm success of PCR. Amplification of PMK unsuccessful. Amplification of all other portions of the plasmid successful.

Monday, June 30

Modified the PCR thermalcycler program (but used the same 50 μL mixture) and ran a gel to check results. All attempts unsuccessful - suspected due to lack of presence of SYBR green in initial mixture.



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