LAB report 12 HOURS( 2 PAGES)

Biol 390-Lab 5 Preparation for Transformation

Objective

· Prepare materials for transformation

· Prepare TAE buffer for electrophoresis in a later lab.

Background

Transformation of bacterial with plasmids is a fundamental technique in genetic engineering. Cells can be made competent to take up plasmids by treatment with divalent cations and heat.

Transformed cells will contain a plasmid that contains a gene that confers resistance to the antibiotic, ampicillin. Therefore, transformed cells can be selected by growth on LB/amp plates. Growth on LB/ara /amp will cause the product to be expressed as the plasmid contains an arabinose promotor

Materials

· LB broth

· LB agar

· Petri dishes

Needed when plates are poured

· Ampicillin (amp) solution sterile – 10 mg/ml stock

· Arabinose (ara) solution sterile – 200 mg/ml stock

For TAE (Tris –acetic acid-EDTA) buffer

· Tris base

· Glacial Acetic acid

· EDTA

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Procedure

Make LB agar

You will need per group

· 1 LB plates

· 2 LB/ amp plates

· 1 LB /amp/ara

How much volume each will you need for the whole class?

How much does a petri dish hold?

Make up separate bottles of LB agar for each set of the plates

Formula for Luria broth.

· Luria broth – 20g/l

· Agar – 15g/l

Heat the broth agar mixture until the agar melts (about 95oC). Your bottles will be autoclaved at 121oC for 20 minutes and stored until 2 days before.

As arabinose or ampicillin can not be autoclaved, sterile solutions of these will be added to the agar after it is re-melted 2 days before the experiment.

TAE Buffer

You need to prepare 200ml of a 50X stock of TAE buffer to use for gel electrophoresis

Recipe for one liter of 50X stock ‘

· Dissolve 242g Tris base in water

· add 57.1mL glacial acetic acid,

· add 18.6g EDTA solution

· bring the final volume up to 1 liter.

This stock solution will be diluted 50:1 with water to make a 1X working solution. The 1X solution will contain 0.4mM Tris, 20mM acetic acid, and 1mM EDTA.