lb – agar medium 1. is taking in a plasmid good or bad for the bacteria? explain. 2. what will...
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LB – Agar medium
1. Is taking in a plasmid good or bad for the bacteria? Explain.
2. What will happen to the bacteria in the two types of growth media? Consider cells with or without plasmid.
LB – Agar +
Ampicillin
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Transformation - Animation
Lab 5a Student Guide
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LB agar: LB = Luria-Bertani (lysogeny) Broth and it contains:10g of tryptone, 5g of yeast extract and 10g of NaCl.
Agar is the crude version of agarose, the gel we used for separation of DNA fragments.
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Background before predictions:Arabinose operon:
Arabinose is a small sugar (monosaccharide), and not very common. When it binds AraC protein, DNA stays open (not looped) and allows RNA polymerase to transcribe the gene downstream.
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See page 6.1
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AraC dimer
arabinose
rfp
Rfpprotein
rfp
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Lab 5a Conclusions 1: Expectations
Lab 5a Conclusions 2:Observations
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pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
rfp 702bp
pARA-R4720 bp
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growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
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Colony isolation and culture
preparing an overnight culture of E. Coli for RFP expression
LB/amp/arabroth
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Accordingly – As a group – make predictions on bacterial growth on 3 different plates:
LB LB-Amp LB-Amp-Arabinose
F- F+F- F+ F+
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Lab Report:
Plate Predict-ions
Reason Results Why
LB
LB-Amp
LB-Amp-Ara
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Sterile technique:To prevent contamination - •Minimize contact with skin. •Shorten open time.•Change tips, spreader. •Use ‘BioHazard’ bags.
“Clam Shell” technique:
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LB-Amp
LB-Amp-Ara
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