#4 – Serial Dilution and Plating of Competent Cells

Reagents/Supplies:

LB plates (4 per group) Competent cells (prepared from previous lab) Micropipettes Micropipette tips 37°C microcentrifuge tubes 5ml vortex cell spreader inoculating turntable flame ethyl alcohol

Overview:

We will determine the concentration of competent cells (cells/ml) by using serial dilutions. A serial dilution can be used to determine the number of any kind of living organism present in a suspension. In this procedure, a small, measured amount of a bacterial culture is diluted into fresh liquid in another tube. A small amount of liquid is taken from this tube and diluted into another fresh tube. This process may be repeated several more times. Equal volumes of liquid are then taken from each of the dilution tubes and plated on culture dishes. The plates are incubated overnight at 37°C. Well separated single colonies will arise on some of the dilution plates. The number of living from the original culture can be calculated from the number of colonies on the dilution plates. The concentration of cells is normally determined by using a spectrophotometer (We will use this method in later labs). However, you should understand how to determine the concentration of cells/ml by serial dilution. This method is often necessary when calibrating a spectrophotometer.

1 Procedure:

A. Serial dilution

1. Obtain a tube of your competent cells from the freezer.

2. Obtain 3 sterile culture tubes.

3. Use a to transfer 4ml of LB medium into the first culture tube. Now use micropipettes to transfer an additional 995μl to the culture tube.  4ml plus 995μl results in a total volume of ______ml.

4. Add 4.5 ml of LB medium into the two other culture tubes.

5. Line the tubes up and label the first tube 1, the second tube 2 and the third tube 3.

6. Using a micropipette transfer 5μl from the prepared competent cells into the first tube of LB medium.  In step 3, you added 4.995ml of LB medium to the first tube. 4.995ml is the same as ______μl.  5μl of competent cells were added to the first tube which contains 4.995ml of LB medium. This results in a total volume of ______μl.  The competent cells were diluted with LB medium. The dilution ratio of the amount of competent cell added to the first tube to the TOTAL VOLUME of solution in the first tube is ______μl:______μl.  Example: If you added 2μl of competent cells to 495μl of LB medium the dilution ratio would be 2:500 or 1:250.  The dilution ratio indicates that the competent cells have been diluted ______x’s.

7. Using the vortex, agitate the tube for 5 seconds.

8. Transfer .5ml of solution from the first tube to the second culture tube and vortex the tube for 5 seconds.  .5ml is the same thing as ______μl.  .5ml of competent cells were add to 4.5 ml of LB medium resulting in a total volume of ______μl.  The dilution ratio for the amount of solution added to the second tube to the TOTAL VOLUME of solution in the second tube is ______μl:______μl.  The dilution ratio indicates that the TOTAL dilution of the competent cells is ______x’s.

2 9. Transfer .5ml of solution from the second tube to the third culture tube and vortex the tube for 5 seconds.  .5ml is the same thing as ______μl.  .5ml of competent cells were add to 4.5 ml of LB medium resulting in a total volume of ______μl.  The dilution ratio for the amount of solution added to the third tube to the TOTAL VOLUME of solution in the third tube is ______μl:______μl.  The dilution ratio indicates that the TOTAL dilution of the competent cells is ______x’s.

B. Plating of diluted cells

1. Obtain four LB plates and label them. Label them so you know which plate corresponds with which culture tube. Note: The first plate will be used for your undiluted competent cells.

2. Go to the one of the cell plating stations which has been set up for you.

3. Take the lid off of your first plate and place it on the inoculating turntable.

4. Using a micropipette, place 100μl of your undiluted competent cells onto the center of your first plate.

5. Take the cell spreader and dip it into the ethyl alcohol.

6. Flame the cell spreader in order to sterilize it.

7. Once the flame has gone out, touch the cell spreader to the agarose gel towards the edge of the plate in order to determine whether it has cooled off enough.

8. Spin the turntable with one hand while using the cell spreader to spread the solution all over the plate. Be certain to cover as much of the plate as possible.

9. Place lid back onto the plate.

10. Repeat steps 3 through 9 for the three remaining plates and the three culture tubes.

11. Incubate the plates overnight at 37°C.

3 Results:

1. Count the colonies on each plate and record your data in the table below.

2. a. 100μl is what fraction of 1ml? ______

b. Therefore, Each plate has _(Fraction)_____ as many colonies on it as are present in each ml of liquid.

c. Therefore, in order to determine the concentration of cells per ml we should multiply the concentration by ______.

3. Determine the concentration (cells/ml) of your undiluted competent cells using the following formula. Complete the table below.

Number of colonies x 10 x factor of dilution

Example: If 22 colonies were observed on the plate which had a 105 dilution.

22 x 10 x 105 = 2.2 x 107 cells/ml

Plate Number of Factor of dilution Concentration (cells/ml) colonies

4 4. Dilution Practice problems.

a. Convert 100mg/ml to μg/μl.

b. convert 100mg/ml to ng/nl.

c. convert 100mg/ml to ng/μl.

d. 10 μl of a sample with a concentration of 100μg/μl is added to 990μl of water. i. What is the dilution ratio? ii. What is the new concentration of the solution?

e. 1 ml of a sample with a concentration of 10mg/ml is added to 99ml of water. i. What is the dilution ratio? ii. what is the new concentration of the solution?

f. 1μl of a sample with a concentration of 10ng/μl is added to 9μl of water. i. what is the dilution ratio? ii. what is the new concentration of the solution?

g. you are given a DNA sample which has a concentration of 2000ng/μl. How would you make 20ng/ul sample?

h. you are given a DNA sample which has a concentration of 250ng/μl. How would you make a 5ng/ul sample?

i. you are given a sample which has a concentration of 1mg/ml. How would you make a 10ng/μl sample? 100ng/μl?

j. you are given a sample which has a concentration of 5 μg/μl. How would you make a sample with a concentration of 100ng/μl?

k. You are given a sample with a concentration of 456mg/ml. How would you make a sample with a concentration of 4μg/ul? 40ng/μl?

l. You are asked to make a solution using three reagents. The concentration of the reagents is as follows: o Reagent 1: 1mg/ml o Reagent 2: 2mg/ml o Reagent 3: 3mg/ml

How would you make a 20μl solution which contains 10ng of reagent 1, 100ng of reagent 2 and 100ng of reagent three?

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