Newcastle University Wetlab/16 September 2008

Tuesday 16th September



 * Plasmid isolated from pGFPrrnB-ncl08 colonies 7 + 11 and pGFPrrnB-BB107 colonies 3 + 5 ON cultures.


 * A Sample of each of the 4 isolated plasmids were restricted using 7.5μl plasmid and 0.5μl each enzyme in 15μl total volume:

- pGFPrrnB-ncl08 restricted with EcoRI + NheI

- pGFPrrnB-BB107 restricted with EcoRI + SpeI

Gel:

Expected band sizes: * Lane 2: ~ 8.4kb and 2.2kb * Lane 3: ~ 8.4kb and 2.2kb * Lane 4: ~ 8.4kb and 3.2kb * lane 5: ~ 8.4kb and 3.2kb

Lane 1: 1kb ladder

Lane 2: pGFPrrnB-ncl08 colony 7 restricted with EcoR1 and Nhe1

Lane 3: pGFPrrnB-ncl08 colony 11 restricted with EcoR1 and Nhe1

Lane 4: pGFPrrnB-BBa_I746107 colony 3 restricted with EcoR1 and Spe1

lane 5: pGFPrrnB-BBa_I746107 colony 5 restricted with EcoR1 and Spe1

As you can see from our gel, all 4 restrictions yielded the expected sized bands, indicating that the ligations had been successful. The remainder of the unrestricted plasmid from pGFPrrnB-ncl08 colony 7 was stored in the -20°C freezer ready to be sent off for sequencing. The two unrestricted pGFPrrnb-BBa_I746107 plasmid samples were then transformed into B.subtilis 168.

Agar plates made:

- pGFPrrnB-BBa_I746107 colony 3 (chloramphenicol)

- pGFPrrnB-BBa_I746107 colony 5 (chloramphenicol)

- B. subtilis 168 (negative control) (chloamphenicol)