LAB report 12 HOURS( 2 PAGES)

Biol 390-Lab 6 Transformation

Objective

· Transform E. coli with the using a plasmid containing Green Florescent Protein (GFP)

Background

Green fluorescent protein from a jellyfish. This protein has been engineered into a plasmid. The plasmid also contains an arabinose promoter that will cause the GFP to be expressed and a gene that codes for a beta-lactamase; an enzyme that breaks down ampicillin.

Plasmid and its genes

Materials

For each group

· E. coli starter plate – streaked for single colonies and 24 hours old

· Agar plates – 1 LB, 2 LB/amp, 1 LB/amp/ara

· Transformation solution – 1 ml color coded

· LB nutrient broth – 1 ml color coded

· Inoculating loops – plastic disposable

· Sterile pipette tips

· Foam tube holder

· Sterile microfuge tubes

· Ice bath

· Stop watch

· Marker

Common work station

· pGLO plasmid

· Heating block – 42oC

· Nitrile gloves

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Prelab Preparation

Two days before

· Add 250ul of LB broth to the lyophilized E. coli using a sterile tip and incubate for 8 to 24 hr at 370C

One day before

· Streak plates for single colonies – incubate 24 to 36 hours. Each group gets one plate

Day of the lab

· Prepare the pGLO plasmid – transfer 250ul of the transformation solution into the lyophilized pGLO vial. Mix by inverting in case there is plasmid DNA attached to the cap.

· Label one tube per group “TR”: aseptically transfer 1000ul to each of the transformation solution.

· Lab one tube per group “LB”: aseptically transfer 1000ul to each of the LB nutrient medium.

Procedure

1. Use gloves during this experiment

2. Clean benches with alcohol – be careful

3. Select two sterile microfuge tubes and label

a. +pGLO

b. –pGLO

4. Place in a rack

5. Open tubes and using a separate sterile pipette tip transfer 250ul of the transformation solution (TR) to each tube.

6. Place tubes in foam rack in the crushed ice bath.

a. Note: make sure the tubes are in contact with the ice. You may have to push them into the foam.

7. Using a sterile disposable loop transfer 2 to 4 E.coli colonies from the agar plate to each tube.

a. NOTE – it important to uses between 2 and 4, 1 to 1.5 mm colonies to maximize the transformation efficiency.

8. Spin the loop in the transformation solution until all the E.coli have been suspended

a. Note there should be no floating chunks.

9. Use a new loop to inoculate the other tube in the same way using other colonies from the plate.

10. Transfer a 10ul aliquot of the pGLO plasmid to the +pGLO tube using a sterile tip and an automatic pipette.

a. NOTE – do not add the plasmid to the –pGLO tube

11. Incubate the tubes on ice for 10 mins once the plasmid has been added.

12. While the tubes are sitting in the ice label your four plates with your name.

a. +pGLO LB/amp

b. +pGLO LB/amp/ara/

c. -pGLO LB/amp

d. –pGLO LB

13. Transfer the tubes into the heating block set at 42oC for exactly 50 seconds. Note – timing is critical

14. After exactly 50 seconds quickly transfer the tubes to ice for 2 minutes.

15. Place the tube back in the microfuge tube rack and add 250ul of LB broth to one tube using a sterile pipette tip. Add 250 ul of broth to the other tube using a new sterile pipette tip.

16. Incubate the tubes for 10 min at room temperature.

17. Gently flick the closed tubes with your finger to mix.

a. Note its important to make sure the tubes are mixed. Pipette 100ul from each tubes as follows:

b. +pGlO tube to:

i. +pGLO LB/amp

ii. +pGLO LB/amp/ara

c. – pGlO tube to:

i. –pGLO LB/amp

ii. –pGLO LB

18. Using a sterile loop spread the suspension gently around the entire surface of the plate. Note this requires very little downward pressure.

19. Turn plates upside down and incubate for 24 hours at 37oC