1. Is taking in a plasmid good or bad for the bacteria? Explain.
What will happen to the bacteria in the two types of growth media? Consider cells with or without plasmid.
LB – Agar medium
LB – Agar + Ampicillin

2. Transformation - Animation
Lab 5a Student Guide

3. 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.

4. 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.

5. See page 6.1

6. AraC dimer
rfp
Rfp
protein
arabinose
rfp

7. Lab 5a Conclusions 1: Expectations
Observations

8. Recombinant plasmid of interest
pARA-R construct
Recombinant plasmid of interest
pARA-R
4720 bp
pARA-R
4720 bp
BamH I
rfp
702bp
rfp 702bp
Hind III 8

9. growth of transformed bacteria on various plates
P+ plates LB
LB/amp
LB/amp/ara
P- plates
No growth LB
LB/amp 9

11. Colony isolation and culture
preparing an overnight culture of E. Coli for RFP expression
Colony isolation and culture
LB/amp/ara
broth 11

12. Accordingly – As a group – make predictions on bacterial growth on 3 different plates:LB
LB-Amp
-Arabinose
LB-Amp

14. Lab Report:
Plate
Predict-ions
Reason
Results
Why LB
LB-Amp
LB-Amp-Ara

15. Sterile technique:
To prevent contamination -
Minimize contact with skin.
Shorten open time.
Change tips, spreader.
Use ‘BioHazard’ bags.
“Clam Shell” technique:

17. LB-Amp
LB-Amp-Ara