|
Printable pdf
Figure 1
1. In vitro phage antibody library screening with antigen (Ag)
2. Preparation of the scFv sub-library, enriched for antigen-specific scFv, from the first round phage infected TG1
Figure 2
3. Construction of yeast scFv-VP16 in vivo expression sub-library for antibody-antigen yeast interaction screening
4. E. coli transformation using electroporation
5. Yeast antibody-antigen interaction screening of the scFv-VP16 library with the antigen “bait”
6. Confirmation of antibody-antigen interaction in yeast clones by b-galactosidase filter assay
7. Plasmid DNA extraction from individual selected yeast colonies
8. Re-testing the scFv-VP16 plasmid using the yeast antibody-antigen interaction assay
9. Characterisation of the antigen-specific intracellular scFv
10. Binding of scFv to antigen by immunodetection assay (Western blot or ELISA).
Figure 3
11. Mammalian luciferase reporter assay for Ag-scFv intracellular interaction
References
|
 |
|
|
LMB Home > PNAC Home > THR Home > IAC Home
4. E. coli transformation using electroporation
Electroporation is an efficient method for transforming E. coli with plasmid, especially for library scale production of recombinants.
Preparation of competent cells for electroporation
a. Inoculate a single colony of E. coli into 5 ml 2 x TY medium. Incubate overnight at 37°C with shaking.
b. Dilute overnight culture into 500 ml of 2 X TY medium in a sterile 2-litre flask. Grow at 37°C until OD600 of 0.6.
c. Chill in an ice-water bath for 10 min and transfer to a chilled centrifuge bottles.
d. Centrifuge for 20 min at 6,000g at 0°C
e. Remove the supernatant and resuspend the pellet in 500 ml of chilled sterile water. Centrifuge again as step 4d.
f. Resuspend in 100ml, spin again as in d and resuspend in 10ml. Spin again and remove the supernatant immediately.
g. Resuspend in 1ml of chilled sterile water. These cells are now ready for transformation.
Transformation by electroporation
h. Using an BioRad Gene PulserTM, the optimal electroporation voltage for DH5a competent cells is 2.5kV in a 0.2cm gap chamber at settings of 200 ohms and 25µF.
i. Add plasmid DNA (maximum volume 3µl) in 50µl of competent cells. Mix gently.
j. Transfer the DNA and cells into a chilled cuvette (gap size 0.2 cm (1) and flick slightly to settle the cells to the bottom. Incubate on ice for 5 min.
k. Place the cuvette into the sample chamber and apply the pulse. Remove the cuvette and immediately add 950µl SOC medium (temperature RT). Transfer to 2ml round bottomed microtube. Incubate at 37°C for 60min with shaking.
l. Plate aliquots of the transformants on TYE plates containing 100µg/ml ampicillin.
Page 5 >
Footnotes
- EQUIBIO Ltd: www.equibio.com Cat. # ECU-102.
|
|