1. A restriction digest on with EcoRI and SpeI was performed. This will cut the promotor out of its vector () which has a RFP reporter gene. We will put (cut with EcoRI and SpeI) in this vector. This way we will get a construct where RFP will be produced dependant on our blue light promotor. We can then compare this with the intensity of RFP produced by .
2. Glycerol stock was made from LigA and . They are stored in the -80°C in S&P F8 67-68 and 69-70.
3. Ecoli strain DB3.1 cells were made competent for electroporesis.
4. Gel electorphoresis and extraction on

NOT PCR

• Electroporation of SAMS+TER ligation (from Tuesday ligation... the one that looked bad....)
• Miniprepped the 4 colonies from SAMS

• Restriction digest of SAMS
• Gel electrophoresis of restriction from ech, fcs, sam5 and sam8
  • Result looks great (woohoo!)
  • Cut and purified from gel

• Used ech and fcs to ligate using 5 μl from ech and 23,5 μl from fcs. There was not enough sam8 for ligation. Redo restriction at some point in the future...
• 4 colonies from sam8 and ech were picked and plated

PCR

• The PCR from yesterday went very well, we had a huge amount of amplified biobrick DNA of the different parts. Agarose gel electrophoresis was used to test if the different pieces of DNA created by PCR had the correct length.

• Amplified DNA was purified and cut with different restriction enzymes. After restriction, DNA was purified again before ligation.

• After the restriction, sam5, sam8 and terminator were ligated in a three-way ligation. The same was done for ech, fcs and terminator. We also started a ligation of just sam5 and sam8 and ech and fcs.

• The Y+K ligation was miniprepped and nanodropped to make later on a stock on glycerol
• We decided to use for virA a puc19-vector in stead of TOPO, puc was digested with Xba1 and Ecor1 and virA was digested with EcoR1 and Spe1. This was done overnight.
• electroporation + plating was done for X+K