BPrac D - Types of experiments

Describe serial dilution test, describe: Perform a serial dilution by a factor of 10 to prepare about 5 cm^3 of 5 concentrations of glucose 

• Eg. Perform a serial dilution by a factor of 10 to prepare about 5 cm^3 of 5 concentrations of glucose 

• Factor of 10 : conc of glucose used for dilution divided by 10 (so you start with 10% , then you continue by using 1% solution to prepare next solution) 

• Limitation : One solution wrong , everything wrong sigh 

Draw serial dilution table

Conc of glucose to be obtained / %	Conc of glucose used for dilution / %	Volume of original stock glucose solution used / cm^3	Volume of distilled water used / cm^3
1 X 10^-1	1	1	9
1	10	1	9
10	10	10	0

Draw gas syringe

Draw displacement of water

Describe t test

• Determine whether there is significant difference between the means of 2 groups

• Data are continuous

Comment and EXPLAIN on what these results show.

• Stomatal density of sun leaves greater than shade leaves and difference is significant 

• Difference in mean will occur by chance in less than 0.05 

• Light is limiting factor in shade 

• CO2 concentration is limiting factor in sun and not limiting

• Higher stomatal density gives a higher rate of uptake of CO2 allowing sun leaves to make use of more available light for photosynthesis

Describe procedure to study transformation

Using:

• +pGLO and -pGLO

• plasmid DNA

• sterile water

• nutrient broth

• + goes on LB/amp and LB/amp/ara plate

• - goes on LB and LB/amp plate

1. Label 2 tubes +pGLO plasmid and -pGLO.

2. Transfer 250l of transformation solution to each tube.

3. Place tubes in ice and ensure they are fully immersed.

4. Remove a colony of bacteria from the E. coli master plate with an inoculation loop and transfer the colony to the +pGLO tube.

5. Twirl the loop between the index finger and the thumb to dislodge the bacteria into the tube.

6. Using a new inoculation loop, repeat steps 4-5 for the -pGLO tube.

7. Transfer 8l of plasmid DNA to the +pGLO tube.

8. Transfer 8l of sterile water to -pGLO plasmid tube.

• Control tube to show that without the plasmid -> bacteria cannot be transformed

9. Mix the contents in the tubes by gently tapping the tubes a few times. 

10. Leave the tubes in ice for 10 minutes.

11. Heat shock the bacterial cells by transferring the tubes into 42C water bath for exactly 50 seconds. Do not shake the tubes.

12. Place tubes back into ice for 2 minutes.

13. Add 250l of LB nutrient broth to each tube and incubate the tubes for 10 minutes at room temperature.

14. Tap tubes gently to mix contents

15. Transfer 100l of solution from the +pGLO tube to a LB/amp plate and a LB/amp/ara plate.

16. Transfer 100l of solution from the -pGLO tube to a LB plate and a LB/amp plate.

17. Spread the solution over the entire surface of the agar plates using a sterile L-shaped spreader. 

18. Use a clean spreader for each plate.

19. Seal the plates with parafilm.

Describe what you would observe on the LB and LB/amp plates for the -pGLO tube. 

• Bacterial lawn on the LB plate but no bacterial colonies on the LB/amp plate 

• Bacteria in the –pGLO tube have not taken up the plasmid -> do not possess the ampicillin resistance gene -> unable to survive in the presence of ampicillin

Identify the plate(s) acting as control(s) and explain the purpose(s) of the plate(s).

• -pGLO LB plate – shows that the bacteria are viable and are able to grow under normal conditions 

• -pGLO LB/amp plate – shows that non-transformed bacteria are unable to grow in the presence of ampicillin

• +pGLO/LB/amp plate – shows that transformed bacteria are able to grow in the presence of ampicillin, but require arabinose as an inducer to express GFP