Growth Curve and Cell Count

Cell count via Hemocytonomer

1. Clean the cover glass mounting support and the coverslip of the hemocytometer with a lens paper and some ethanol.
   Note: Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid.

2. Place the coverslip over the counting surface prior to putting on the cell suspension.

3. Introduce the suspension into one of the V-shaped wells with a pasteur or other type of pipet.
   Note: The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered.

4. Place the counting chamber on the microscope stage and bring the counting grid is into focus at low power.

Growth Curve

1. With the use of a hemocytometer, count the number of cells present in a small sample of your culture

2. With this number, calculate the cell density (cells/ml) of your culture

3. For the same culture, take cell count measurements on a regular basis (every 5 hours) for an entire week

4. Record all the data you collect and the time at which you took the measurement!

5. Once the week is over, plot cell density versus time in order to obtain a growth curve

6. You must be able to see the different growth phases the species goes through (log, stationary)

Antibiotic Spot Test

Gibson Assembly

1. The DNA you wish to be assembled and the Gibson Master Mix should be combined with a volumetric ratio of 1:3 in a PCR tube.

   1. Note: we need 300 femtomoles of each part for the reaction
   2. The total volume can be from 20-50µl.

2. The PCR tube should then be incubated for 1 hour at 50°C

Gibson Master Mix:
Taq Ligase (40 u/µl) -- 50 µl
5x isothermal buffer -- 100 µl
T5 exonuclease (1u/µl) -- 2 µl
Phusion polymerase – 6.25 µl
Nuclease-free water – 216.75 µl
Total Volume → 375 µl

5x Isothermal buffer: 25% PEG-8000 – 0.75 g
500 mM Tris-HCl pH 7.5 -- 1500 µl
50mM MgCl2 -- 75 µl
50mM DTT -- 150 µl
1 mM dATP – 30 µl
1 mM dTTP -- 30 µl
1 mM dCTP -- 30 µl
1 mM dGTP -- 30 µl
5mM NAD -- 300 µl
Nuclease-free water – remainder
Total Volume → 3000 µl