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From 2014hs.igem.org

Methods

Preparing competent cells

The bacteria were grown to log (or exponential) phase and 10mL of bacteria were

aliquoted into 15 mL sterile plastic tubes. cells were pelleted using a tabletop

centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. The supernatant was poured

off into liquid waste container. the tube was tapped to loosen the cell pellet, and d 5

mL of cold CaCL2 solution were added to the pellet. The cell solution was incubated

on ice for 20 minutes. After incubation, the cells were spun for 5 minutes at 5000 x

g (2500 rpm). The supernatant was poured off into the liquid waste container. One

mL of cold CaCl2 solution was added to the cells and the cells were resuspended

Rehydration of RFP and Alcohol Acetyltransferase I

Pipetted 10uL of dH2O into the well of corresponding Biobrick-standard part. Pipetted up and down, then let sit for 5 minutes. Tranformed 5 uL of the re-suspended DNA into desired cells, plate, and grew overnight.

Transformation of RFP and Alcohol Acetyltransferase I

Labeled tubes as Transformation Control, Ligation: New Part, and Ligation Control. Added 5uL of RFP Control DNA into Transformation Control. Added 2uL of New Part ligation product into Ligation: New Part. Added 2uL of the RFP Control ligation product into the Ligation Control. Put tubes on ice to pre-chill, and thawed one competent cell tube on ice. Pipetted 50uL of competent cells into each 2.0mL micro-centrifuge tube. Incubated DNA and cell mixtures on ice for 30 minutes. During incubation, preheated waterbath to 42˚C. Placed tubes into waterbath for 60 seconds, then incubated on ice for 5 minutes. Added 200uL of SOC media to each tube. Incubated tubes at 37˚C for 2 hours. Pipetted 200uL of each tube onto respective plates. Spread over surface using glass beads. Incubated plates facing down for 37˚C for 12-14 hours.

Extraction of RFP and Alcohol Acetyltransferase I

Spun down two culture tubes for 3 minutes at 8000 rpm. Poured supernatant into biological waste container. Pipetted 250uL of Buffer P1 into each 14mL culture tube, and resuspended pellet. Labeled 1.7mL micro-centrifuge tubes with respective part names, and transferred resuspended parts in. Pipetted 250uL of Buffer P2 into each micro-centrifuge tube. Flipped tubes to mix solution. Pipetted 350uL of Buffer N3 into each tube. Flipped tubes to mix solution. Spun down tubes in micro-centrifuge for 10 minutes at 13,000 rpm. Labeled spin columns for each tube, and pipetted supernatant to the respective spin columns. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 500uL of Buffer PB into each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 750uL of Buffer PE to each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Spun down spin columns at 13,000 rpm for 1 minute to remove remaining buffer. Labeled sterile 1.7mL micro-centrifuge tubes with part names. Transferred the appropriate filter tube to the respective tubes. Pipetted 50uL of dH2O to each filter tube. Let samples sit for 1 minute, then spun down tubes at 13,000 rpm for 1 minute. Stored micro-centrifuge tubes at 20˚C.

Digestion of RFP and Alcohol Acetyltransferase I

Thawed NEB Buffer 2 and BSA in room temperature water. Labeled four 0.6mL tubes: Part A, Part B, pSB1C3, and RFP Control. Added 500ng of DNA to appropriate tube. Added distilled water to tubes for a total volume for 42.5uL in each tube. Pipetted 5uL of Buffer 2 to each tube. Pipetted 0.5uL of BSA to each tube. In Part A tube, added 1 uL of EcoRI enzyme and 1uL of SpeI enzyme. In Part B tube, added 1uL of XbaI enzyme, and 1uL of PstI enzyme. In the pSB1C3 tube, added 1uL of EcoRI enzyme, and 1uL of PstI enzyme. In the RFP Control tube, added 1uL of EcoRI enzyme, 1uL of PstI enzyme. Mixed solution by pipetting up and down, and spun for 5 seconds in micro-centrifuge to collect mixture at the bottom of the tube. Incubated the restriction digests at 37˚C for 30 minutes, then 80˚C for 20 minutes. Stored at -20˚C.

Ligation of RFP, Alcohol Acetyltransferase I, and PSB1C3

Thawed T4 DNA Ligase Reaction Buffer. Labeled 0.6mL as New Part. Added 2uL of pSB1C3 linearized plasmid backbone digest. Added 3.3uL from Part A digest. Added 3.9uL from Part B digest. Added 1uL of T4 DNA Ligase Reaction Buffer. Added 0.5uL of T4 DNA Ligase. Mixed solution by pipetting , and centrifuged for 5 seconds. Labeled another 0.6mL tube as Ligation Control. Added 2uL from RFP Control digest. Added 6.5uL of distilled water. Added 1uL of T4 DNA Ligase Reaction Buffer. Added 0.5uL of T4 DNA Ligase. Mixed solution by pipetting, and centrifuged for 5 seconds. Incubated both tubes at 16˚C for 30 minutes, then at 80˚C for 20 minutes. Stored at -20˚C.