A: How can you determine if the bacteria have picked up your vector (plasmid) DNA?

A; How do we know we got the desired gene inside the bacteria?

If you start with bacteria that is ampicillin sensitive and have a plasmid enter in with an amp-resistant gene, the bacteria will be amp-resistant and grow on mediums with ampicillin on it. Bacteria that did NOT pick up the plasmid will die on the ampicillin plate

B: How can you determine if you have inserted any DNA in to your vector (plasmid) DNA?

Failed insertion of foreign DNA in the lacZ gene: the protein B-galactosidase substrate will produce blue colonies, so this means the LacZ gene is functional
Successful insertion: Only white colonies will grow due to lack of X-Gal (substrate from B-gal that makes colonies blue, we WANT this to show the GMO)

C: how can you determine which clone contains the DNA of interest to you?

1: Take all the white colonies into a plate and use a nitrocellulose filter to make a copy of the location of each colony, keep the original plate for comparison

2: Use detergent to lyse the bacteria so the nitrocellulose can access the DNA in the bacterial cells

3: Treat the nitrocellulose filter with NaOH (base!) to make the DNA single stranded (ask if we need to know how)

4: Add a probe with fluorescence to identify where it binded in the cells; the single-stranded probe will stick onto the single stranded DNA with desired gene

5: Shine light to the probe, wash filter to remove the probe and then compare with original plate to colonies to look into (ask)

PCR next

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