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.

• • • • 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