John Kosta

3/20/18

4*

Lab Write Up

Purpose/Problem/Question

The Purpose of this lab is to discover the effects of the gfp gene on bacteria cells.

Hypothesis

If we put the pGlo plasmid into E. coli bacteria, then both LB plates will show growth but not
glow, LB + Amp + DNA will show growth but not glow, and LB + Amp + ara + DNA will both
grow and glow; all other plates will not show growth.

Procedure

Before beginning the lab sanitize all surfaces on which the lab will be performed. Wash your

hands before the lab and after completion.

Materials:

- E. coli starter plate

- Poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara)

- Transformation solution

- LB nutrient broth

- Inoculation loops

- Pipette

- Foam microcentrifuge tube holder/float

- crushed ice (not cubed ice)

- Marking pen

- Microcentrifuge tubes
- Rehydrated pGLO plasmid

- 42°C water bath and thermometer

- UV Light

- 37°C incubator

- adjustable volume micropipette 2–20 µl

- micropipette tips

Procedure

1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your

group’s name. Place them in the foam tube rack.

2. Open the tubes and, using a sterile transfer pipet, transfer 250 µl of transformation

solution (CaCl2) into each tube.

3. Place the tubes on ice.

4. Use a sterile loop to pick up 2–4 large colonies of bacteria from your starter plate. It is

important to take individual colonies (not a swab of bacteria from the dense portion of the

plate), since the bacteria must be actively growing to achieve high transformation

efficiency. Pick up the +pGLO tube and immerse the loop into the transformation

solution at the bottom of the tube. Spin the loop until the entire colony is dispersed in the

transformation solution. Place the tube back in the ice. Using a new sterile loop, repeat

for the -pGLO tube.

5. Pipet 10 µl of pGLO plasmid into the +pGLO tube & mix. Do not add plasmid DNA to

the - pGLO tube. Close both the +pGLO and -pGLO tubes and return them to the ice.

6. Incubate the tubes on ice for 10 min.

 7. While the tubes are sitting on ice, label your five LB nutrient agar plates on the

bottom(not the lid) as LB/amp +pGLO, LB/amp/ara +pGLO, LB/amp -pGLO, LB

+pGLOand LB -pGLO.

8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO

tubes into the water bath, set at 42o C, for exactly 50 sec. Make sure to push the tubes all

the way down in the rack so the bottom of the tubes stick out and make contact with the

warm water. When the 50 sec are done, place both tubes back on ice. Incubate tubes on

ice for 2 min.

9. Remove the rack containing the tubes from the ice and place on the bench top.Open a

tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to the tube and

reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 min

at room temperature.

10. Tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube,

pipet 100 µl of the transformation and control suspensions onto the appropriate plates.

11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of

the agar by quickly skating the flat surface of a new sterile loop back and forth across the

plate surface.

12. Stack up your plates and tape them together. Put your group name and class period on the

bottom of the stack and place the stack upside down in the 37°C incubator until the next

day.

Data/Observation
Observations: During this lab we made observations about what was happening in the lad. When

making the gel agar plates, i observed the smell coming from the liquid to be sickening. The

consistency of the agar was gelatin like but much firmer. of this lab

+DNA LB plate. This plate didn’t grow or glow, going against our hypothesis our hypothesis.

Here we have the -DNA LB plate. The bacteria received growth but didn’t glow.

Above are the results for the +DNA LB Amp plate which grew but didn’t glow.
Above is the -DNA for the LB+Amp which experienced no growth or glow.

The above picture shows the +DNA for the LB+Amp+Ara where the bacteria did not glow or
grow.

Analysis/Discussion

Each group in the class tested five plates with positive plates containing pglo and negative plates

containing no pglo. If the procedure was done correctly then the correct results would be as

follows:

LB LB+Ampicillin LB+Ampicillin
+Arabinose

DNA+ Growth/No Glow Growth/No Glow Growth/Glow

DNA- Growth/No Glow No Growth/No Glow

LB is the base gelatin like substance that acts as an environment for bacteria to grow. This is

why growth should be present in both the positive and negative LB plates. The ampicillin kills

bacteria which is why there should be no growth in its negative plate. But in its positive plate, the

DNA is resistant to the ampicillin letting the bacteria grow. The arabinose allows the GFP gene
to be correctly expressed allowing the bacteria to glow. The data for my groups lab can be found

in the table below:

LB LB+Ampicillin LB+Ampicillin
+Arabinose

DNA+ No Growth/No Glow Growth/No Glow No Growth/No Glow

DNA- Growth/No Glow No Growth/No Glow

This data does not match with the correct results meaning there were mistakes throughout the

lab. The Lb should have grown in both positive and negative. The LB and ampicillin plates were

correct. The arabinose plate should have grown and glowed, however it did not. This goes

against the hypothesis that corresponds with the correct results table. It is incorrect for the

reasons stated above in relation to our results.

Some errors may have included not putting our solutions onto ice for long enough or fast

enough. This may have affected how the agar was produced. Another mistake during the lab

could have been not properly heating the solutions in the water heater bath. This could have

affected how the DNA was shocked and it may have been done improperly because the time of

heating was very precise and using a clock across a lab may not have been the best timing

technique. One other error that may have occurred was the incorrect sterilization of the loop to

pick up the bacteria colonies. This could have contaminated the bacteria skewing the results of

the lab.

One to improve the lab is making the procedure simpler. Throughout the lab my group and i

struggled to understand what each step of the procedure was explaining. It would be helpful to

keep the procedure to only the essential information as not to overload people with steps to go

through. This would have helped a lot throughout the lab especially in my group. Another issue
was that we couldn’t see specific mistakes that happened in the lab. The only way to get better

results is to redo the lab but you can never pinpoint a single mistake that was made. This is

frustrating as groups perform this lab. My overall discovery in this lab.

Conclusion

My main discovery from this lab was that there were many mistakes that affected the final

outcome. This lab let us use E Coli cells to discover how they are affected by pGLO and

arabinose. We tested five plated of bacteria for their growth and ability to glow under a

fluorescent light. Some evidence for the mistakes throughout the lab were that the results were

not correct. Other pieces of evidence are explained in the analysis. Also the following tables

show the differences in the results of the lab.

Correct:

LB LB+Ampicillin LB+Ampicillin
+Arabinose

DNA+ Growth/No Glow Growth/No Glow Growth/Glow

DNA- Growth/No Glow No Growth/No Glow

Ours:

LB LB+Ampicillin LB+Ampicillin
+Arabinose

DNA+ No Growth/No Glow Growth/No Glow No Growth/No Glow

DNA- Growth/No Glow No Growth/No Glow

This proves that there was at least one error that occurred in our lab. In order to see the correct

results we can perform the lab again.