Team:Bio Without Borders/Notebook

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Notebook

Week 1 (June 4-8)

•Made competent cells and aliquoted them into 25 1.5 mL tubes (50 microL each)

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. They grow better in 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 aliquoted 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.

•Filled out first draft of safety form

•Went to Brighton Beach to examine horseshoe crabs' habitat and found a part of the carcass

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 pSB1C3 backbone amplification PCR using Q5.

•Ran gel of pSB1C3 j04450 insert with Q5 PCR to confirm size.

•Amplification of pSB1C3 j04450 insert with Phusion PCR (because Q5 did not work). Confirmed size with gel electrophoresis.

•Plasmid prep of pSB1A3

•Digest of amplified pSB1C3 j04450 insert (Phusion PCR).

•Amplified linearized backbone from pSB1C3 and pSB1A3 with Phusion PCR

•Ran a gel for pSB1A3 and pSB1C3 plasmid prep from day before to check band size

•Purified (accidentally-amplified with Phusion) PSB1C3 j04450 insert, as well as the circular (made into linear) pSB1A3 and pSB1C3 backbones with PCR clean-up

Week 5 (July 2-9)

•Went to Jones Beach to examine horseshoe crabs' habitat and found a carcass

•Worked on GoFundMe campaign page

•Digested pSB1K3 backbone, ligated it with the J04450 insert (from purified J04450 insert from 6/27), and transformed cells for Kanamycin resistance & plated them

•Ran cPCR for transformed kanamycin resistant colonies

•Coded format for iGEM website

•Inoculated LB broth with transformed kanamycin resistant colonies