Big goal: Set up the well plates for the experiment comparing WT and 0948 KO genotypes on phage infection and create cultures with increased kanamycin to attempt total knockout
Materials:
Micropipette 20
ClarioStar Well Reader
Disposable pipettes 5 ml + 10 ml + 1 ml
96 plate wells
Test tubes
Test tube racks
Pipette controller gun
Fume hood
Gloves
Pro 99
Synechococcus 7803(The sample we have prepared have fluorescent dyes already)
Synechococcus 0948KO(The sample we have prepared have fluorescent dyes already)
Phages S-SMI/S-SCSM1o/1p/3a/4a
Kanamycin
Procedure number 1: Comparing syn 7803 and 0948KO
Turn on UV in fume hood one hour early
Put on gloves
Get synechococcus 7803 and 0948KO
Sanitize fume hood+hands+all materials(keep phages in the fridge)
Transfer cyanobacteria into new culture(done on weekly basis)
Transfer pro99 into small tube to avoid contamination
Add pro 99 and old culture using pipette controller gun and pipette tips so that each of the three new tubes are the same pinkish color
Pipette 200 microliters of each culture into the well plates
Use Clariostar well reader fluorescence to measure density
Use the 7803 and 0948 KO cultures closest in density
Get phages out of the freezer
Pipette into well plates
Pipette 200 microliters of pro 99 on the outer layer of the well plates
Pipette 190 microliters of pro99 into the rest of the well plates
Add 20 microliters WT on left and KO on right(10:1 dilution but the phages absorb)
Add the phages(one phage per row) control(no phage), 1o, 1p, 3a, 4a
