Adam Crego's Laboratory Notebook

02/06/08 Transformation of DNA
Name: control, Part BBa_J45219

Group 1:

Parts: (B0032) --> ATF1 (J45014) --> (B0010) --> (B0012)

Library: iGEM 2007, Well: 3J,  Plate: iGEM 2007 Parts Kit Plate 3,  Plasmid: pSB1AK3,  Cell: V1009,  Available

Transformation of Recombinant DNA

 * 1) ) Obtain specific plate from refrigerator. A number and a letter.  ex. 3J
 * 2) ) Set to 8 or 9 microL on pipette
 * 3) ) Inject distilled water into the well (pipette up and down a few times) and mix.
 * 4) ) Water should be orange from DNA.

•	Making lots of DNA - Amplifying using PCR •	Save some cells (DNA) in freezer at -80 degrees C
 * Only need 4 microL for a transformation (part/date/initials on tube)

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