Holmesglen: HSB:hg 29-Oct-2015 U:\HSCS\HSB\Programs - Courses\LAB TECH\Course Delivery & assessment\CERT IV IN LAB TECHNIQUES\2015\MSL974006A Perform Biological Procedures\Alternate pGLO SOP.docx

MSL40109 Certificate IV in Laboratory Techniques MSL974006A Perform Biological Procedures MSL933001A Maintain the laboratory/field workplace fit for purpose

Alternate pGLO SOP

1. You will be given a McCarney bottle with E. coli in LB broth.

2. Transfer the contents into a plastic centrifuge tube (blue topped falcon tube) and centrifuge. Discard the supernatant into a safe container for decontamination.

3. Resuspend the pellet in 100µl of 100mM ice cold CaCl2 (pH

4. Incubate on ice for 10 min

5. Re centrifuge and discard the supernatant in a safe container

6. Resuspend the cells in 200µl 10mM CaCl2

7. Take 100µl of this suspension and add to a sterile eppi tube containing 5µl

plasmid DNA, and 100µl into another sterile eppi tube containing 5µl sterile dH2O

Cells in 200µl 10mM CaCl2

100µl 100µl

Add 5µl pGLO plasmid Add 5µl sterile dH2O

8. Incubate on ice for 10 min (or longer if possible)

9. Heat shock at 42oC for 50 seconds

2 Holmesglen: CLS HSB 19-Jan-15 H:\AST\BIO\CERT IV PATHOLOGY\2014\HLTPAT306D Perform Blood Collection HLTPAT404D Collect Pathology Specimens other than Blood cluster\Student

Notes\Student notes week two on.docx

10. Place cells back on ice for 2 min

11. Transfer cells to 900 µl LB broth and incubate in the 37oC shaking incubator

for 1 hour. This is a recovery step which allows time for the β lactamase production to allow ampicillin resistance)

12. Plate 100µl of this suspension onto the relevant agar plates and spread with a

spreader

Biotechnology Explorer TM

pGLOTM

Bacterial Transformation Kit pp 15

13. Incubate at 37oC overnight.