Question

Biology Lah 10. (20) You are setting up an experiment to electroporate AX2 cells with a new vector. The vector has a Belomycin resistance marker gene. The literature tells you that Bleomycin is effective on dicty in a concentration range of 1-50 μg/ml in HL-5 media and cells die after 1-5 days. Your boss tells you that you need to determine the best concentration of Bleomycin to kill Dicty before you begin your electroporation experiment. How would you set up such an experiment using a 10 ml culture of Ax2 at 1x 106 cells/ml and the lab stock of Bleomycin which is at a concentration of Img/ul. (Hint: think about testing several difference concentrations of drug) A good way to present this answer would be as a lab protocol. Make sure that you indicate how you will determine the results of the experiment

Answer 1

We have 10ml culture of   Ax2 with 1*10⁶ cells/ml

We have Bleomycin of 1mg/μl

Now we have to find the minimum concentration of antibiotic required to kill non-transfected (plasmid DNA) cells.

• Take a 6 well plate and add 2ml HL-5 media to every well.
• Now add 100μl of AX2 culture with 1*10⁶ cells/ ml cells and allow it to grow for one day
• Remove the media and add fresh media and bleomycin in the given quantity for every well
• Concentration of stock antibiotic is 1mg/μl= 1000 μg/ μl
• Take 1 μl of this stock and add 999 μl of media now the concentration of new stock is 1000 μg/ 1000μl=1μg/ 1μl

We know optimum concentration is between 1-5 0 μg/ml

1^st well

Let us keep the concentration of antibiotic as 1 g/ml

So in 2 ml media we need 2 g of antibiotic to make a final concentration of 1 g/ml

Add 2 l new antibiotic stock and 1998 l of media to the first well

2^nd well

Let us keep the concentration of antibiotic as 10 g/ml

So in 2 ml media we need 20 g of antibiotic to make a final concentration of 10 g/ml

Add 20 l new antibiotic stock and 1980l of media to the first well

3^rd well

Let us keep the concentration of antibiotic as 20 g/ml

So in 2 ml media we need 40 g of antibiotic to make a final concentration of 20 g/ml

Add 40 l new antibiotic stock and 1960l of media to the first well

4^th well

Let us keep the concentration of antibiotic as 30 g/ml

So in 2 ml media we need 60 g of antibiotic to make a final concentration of 30 g/ml

Add 60 l new antibiotic stock and 1940 l of media to the first well

5^th well

Let us keep the concentration of antibiotic as 40 g/ml

So in 2 ml media we need 80 g of antibiotic to make a final concentration of 40 g/ml

Add 80 l new antibiotic stock and 1920 l of media to the first well

6^th well

Let us keep the concentration of antibiotic as 50 g/ml

So in 2 ml media we need 100 g of antibiotic to make a final concentration of 50 g/ml

Add 100 l new antibiotic stock and 1900 l of media to the first well

• Keep the cell for 5 days. Every day check the cells under microscope to see the cell death. Cell will round up as they die

Here we can find the range of concentration in which the cell death is maximum. Let the concentration we get here is 20 g/ml .

Then do the same experiment for the concentration near to 20 g/ml like 15 g/ml, 18 g/ml, 22 g/ml, 25 g/ml

Then you will get accurate result