Adam Crego's Laboratory Notebook

02/06/08 Transformation of DNA

Name: control, Part BBa_J45219 and BBa_R0011R

Group 1:

Parts: (B0032) --> ATF1 (J45014) --> (B0010) --> (B0012) Library Well Plate Plasmid Cell iGEM 2007 3J iGEM 2007 Parts Kit Plate 3 pSB1AK3 V1009 Available

Transformation of Recombinant DNA

1. .) Obtain specific plate from refrigerator. A number and a letter. ex. 3J

1. .) Set to 8 or 9 microL on pipette

1. .) Inject distilled water into the well (pipette up and down a few times) and mix.

1. .) Water should be orange from DNA.

• • • • Only need 4 microL for a transformation (part/date/initials on tube)

• Making lots of DNA - Amplifying using PCR • Save some cells (DNA) in freezer at -80 degrees C

Transformations:

1.) If using DNA from Registry, dilute with 9 microL of H20. 2.) Label 2 tubes w/ (part/date/name) 3.) Add 100 microL competent cells (strain of E. coli : JM109, company Promega, catalog #L2001) in each tube 4.) Add 4 microL of Registry DNA into only one tube 5.) Place in ice bath for 30 minutes 6.) Heat shock at 42 degrees C for 45-60 seconds 7.) Place in ice bath for 2 minutes 8.) Add 900 microL ice cold antibiotic free LB to cells 9.) Incubate at 37 degrees C for 90 minutes in a shaking incubator 10.) Dilute as desired, make necessary controls, and plate cells (200 microL per plate) 11.) Incubate upside down at 37 degrees C for 18-24 hours, then refrigerate

3 Plates: 1.) Control: 100 microL LB/cell 2.) 2 test plates 1:1 100 micro LB/cells 1:100 1 microL cells to 99 microL H20