What size fragments would you expect when cutting the plasmid with the two enzymes used

DO NOT DO RESULT SECTION
This lab report will cover the transformation of E.coli  and the restriction digest of the plasmid (labs 5 to 8).
Use the Standard lab report format and include the following
– Overall objective
– Background to what you were trying to do
– Methods – used to create reagents and used for the transformation, plasmid isolation, restriction digest and agarose gels.  Create sub section for each
Transformation
– What was the selectable marker?
– How do you know you successfully transformed the cells with pGlo
– What was the most critical step in the transformation process
Plasmid preparation
– Why does only the E. coli grown in ara/amp fluoresce
Restriction digest
– What size fragments would you expect when cutting the plasmid with the two enzymes used?
 
– Are the results of the plasmid what you expected.
– Are the two fragments the sizes you expected ?
Biol 390-Lab 5 Preparation for Transformation

2

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.
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