Team:Bio Without Borders/Notebook

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Notebook

Week 1 (June 4-8)

•Made competent cells

Week 2 (June 11-15)

•Testing competent cells using iGem DNA transformation samples.

• Tested to see if they grew better on a freezer block versus an ice bath.

• Ran colony PCR and plated bacteria on antimicrobial plate.

• cPCR, chloramphenicol, and culturing protocols

• Ran 1% agarose gel for cPCR

• Plasmid prep of PSB1C3

Week 3 (June 18-22)

•Made chloramphenicol LB plates

•Restriction enzyme digest of iGem plasmid pSB1C3 using EcoR1 and Pst1

•Made digest master mix; ran an ezyme digest of iGem plasmids pSB1A3 and pSB1K3 Performed ligation of

pSB1A3 and pSB1K3; transformed pSB1A3 in competent cells on ampicillin plate

•Created a 50mg/mL kanamycin solution and then alloquoted it to about 80 1.5mL tubes.

•Inoculated LB with ampicillin colonies (with insert).

•Streaked ampicillin (1A3) colony onto new plates.

•Conducted cPCR for 1A3 colonies with insert.

Week 4 (June 25-29)

•Conducted cPCR for PSB1K3.

•Ran 1% agarose gel of pSB1A3 with the insert (colonies that were re-streaked).

•Ran 1% agarose gel of pSB1K3 with insert.

•Cleaned laboratory material.

•Set up PCR using Q5 mastermix.

•Amplification of pSB1C3 plasmid backbone.

•Plasmid prep of PSB1A3

•Digest of linearized PSB1C3

•Set up phusion PCR and gel electrophoresis of PSB1A3, using primers VR and VF2.

•TMR!!! Amplification of pSB1C3 and pSB1A3, ran it through PCR cleanup and digested with EcoR1 and Pst1.