Team:Charlottesville RS/Notebook/material

From 2014hs.igem.org

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(Rehydration of RFP and Iso)
(Transformation of RFP and Iso)
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===Transformation of RFP and Iso===
===Transformation of RFP and Iso===
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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 Iso===
===Extraction of RFP and Iso===

Revision as of 13:47, 2 May 2014

Material

Buffers & Solution

Kits

Marker

Enzymes

Plasmidvectors

Synthetic oligonucleotides

Phages

Bacteria

Bacteria Growth Media

Methods

Preparing competent cells

Before lab, bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. Pelleted cells using tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. Supernatant was poured off into liquid waste container. Tapped the tube to loosen cell pellet, and pipetted 5 mL of cold CaCL2 solution. Incubated tube on ice for 20 minutes. After incubation, spun cells for 5 minutes at 5000 x g (2500 rpm). Poured off supernatant into liquid waste container. Used pipette to add 1 mL of cold CaCl2 solution and resuspended cells.

Rehydration of RFP and Iso

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 Iso

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 Iso

Digestion of RFP and Iso

Digestion of PSB1C3

Ligation of RFP, Iso, and PSB1C3

Transformation of RFP, Iso, and PSB1C3