Week 4

August 1-

Miniprep

Observations: i2.11.6 grew, i2.10.7 no growth

Making glycerol stock

Dissolving and diluting primers (10 pmol/ µL)

PCR and gel electrophoresis and purification

At this stage we have both the FOFr and CheZ* with XbaI and SteI restriction sites. Our next goal will be to insert each part into a vector backbone so they will be ready for addition of the ToxR and Ctx genes. Once these two fusion products are made, we hope to transform and test whether local motion can be inhibited with CheZ and the receptor combo

August 2-

Gel electrophoresis and purification

Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify

Ligation at 16 degrees Celsius overnight

We added both CheZ* and FGFR each into its own vector and if the ligation just performed works we will have 2 new biobricks. The phosphorylation was done to prevent ligase from reannealing the X and S site on the vector. This means only the phosphate on the insert can be used to connect the 2 DNA sequences.

August 3-

Transformation of FOFR and CheZ*, DH5α, 50 µL 2 µL DNA at 37 degrees Celsius

August 4-

Incubation in 3 mL and chlora at 37 degrees Celsius and 220 rpm

Plating CheZ (K283006) and ToxR (J07009)

Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify

August 5-

Making glycerol stock

Miniprep

Incubation in 3 mL LB carb at 37 degrees Celsius and 220 rpm overnight

Test digestion ( 1 hour and 30 minutes)- about 200 ng of DNA

Gel electrophoresis

Observations: i2.15.16 looks positive, the rest are negative

August 6

Glycerol stock and miniprep