1. Is taking in a
plasmid good or
LB – Agar
bad for the
medium
bacteria? Explain.
2. What will happen
to the bacteria in
the two types of
LB – Agar
growth media?
+
Consider cells with Ampicillin
or without plasmid.
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
rfp
arabinose
rfp
Rfp
protein
Lab 5a Conclusions 1:
Expectations
Lab 5a Conclusions 2:
Observations
pARA-R construct
Recombinant plasmid of interest
pARA-R
pARA-R
4720 bp
4720 bp
rfp
rfp 702bp
702bp
growth of transformed bacteria on various plates
P+ plates
LB
LB/amp
P- plates
No growth
LB
LB/amp
LB/amp/ara
preparing an overnight culture of E. Coli for RFP expression
Colony isolation and culture
LB/amp/ara
broth
Accordingly – As a group –
make predictions on bacterial
growth on 3 different plates:
F- F+
LB
F- F+
LB-Amp
F+
LB-Amp
-Arabinose
Lab Report:
Plate
LB
LBAmp
LBAmpAra
Predict Reason
-ions
Results Why
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