1. The plates with ligation A (blp + GFP) were fetched from the blue light installation. There was no GFP signal. The following actions will be taken:
   • The plasmids will be purified from the colonies and will be sequenced using primer 2260.
   • Next time, we will probably put them in liquid cultures under the LED's while shaking gently. Also, other parameters that need to be considered are being researched.
   • They will not be exposed to the light as long anymore. We decided that 1h will be more than enough.
   • Possible bleaching?
2. A colony from all three plates with the LigA-construct was taken and ented in liquid culture. This was needed to check whether the colonies were still alive.
   • culture 13/08
   • culture 14/08 (1)
   • culture 14/08 (2)
3. By 5pm, one of the liquid cultures had grown and was miniprepped. 20μl was sent for sequencing together with 5μM of primer 2260.

4. Two electroporations were performed and plated on LB medium. One with LigC ( + ) and one with DNA.

• Miniprep of fluid cultures

Nanodrop results:

• Restriction:

-We performed 2 simultaneous digests: one to verify the length of the product (Restriction2Test) using SAMS II and EF III and another to prepare for ligation (Restriction4Real) with promotor and terminator using SAMS III and EF II

-We cut with the 2 enzymes separately, allowing a one hour incubation in between. EF: E/S SAMS: E/S

-Restriction volumes:

Restriction2Test

Restriction4Real

• Terminator was also digested

Terminator: E/X

• Restriction2Test: Agarose gel electrophoresis to validate if the test samples have the correct length. Signals appeared that could not be explained

• PCR for the second mutation of R was done
• Electroporation of B and G after the ligation yesterday
• PCR was done for controle of W to check if virA was inserted into the cell. The picture made after gelelectrophorsis showed a wrong result