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
Transformation - Animation
Lab 5a Student Guide
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.
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.
See page 6.1
AraC dimer
arabinose
rfp
Rfpprotein
rfp
Lab 5a Conclusions 1: Expectations
Lab 5a Conclusions 2:Observations
pARA-R construct
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
rfp 702bp
pARA-R4720 bp
growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
Colony isolation and culture
preparing an overnight culture of E. Coli for RFP expression
LB/amp/arabroth
Accordingly – As a group – make predictions on bacterial growth on 3 different plates:
LB LB-Amp LB-Amp-Arabinose
F- F+F- F+ F+
Lab Report:
Plate Predict-ions
Reason Results Why
LB
LB-Amp
LB-Amp-Ara
Sterile technique:To prevent contamination - •Minimize contact with skin. •Shorten open time.•Change tips, spreader. •Use ‘BioHazard’ bags.
“Clam Shell” technique:
LB-Amp
LB-Amp-Ara