Team:Aberdeen Scotland/Transformation

Transformation

The following are different transformation methods used as part of our project

Transformation of TSS competent cells

1. Thaw 100µl of TSS/cells on ice
2. Add 1µl of biobrick DNA, pipette gently to mix(100pg – 1 ng of plasmid in a volume of 1-2µl)
3. Incubate on ice for 30 minutes
4. Add 0.9ml SOC at 4°C
5. Incubate for 1 hour at 37°C in shaker
6. Spread 100 µl onto agar plate, containing appropriate antibiotic (see note ii.)
7. Centrifuge remainder of cells at 200rpm / 5 min in a microfuge
8. Remove all except 50 µl of supernatant: resuspend gently with a pipette
9. Plate on a fresh agar plate, containing antibiotic
10. Invert plates and incubate overnight at 37°C


Notes; i) Antibiotic concentrations in LB:
Ampicillin - 100 µg ml-1
Tetracycline - 10 µg ml-1 in ethanol
Chloramphenicol - 30 µg ml-1 in ethanol
Kanamycin - 30 µg ml-1 in dH2O

ii) Antibiotic concentrations in LB-agar:
Ampicillin - 100 µg ml-1
Tetracycline - 5 µg ml-1 in ethanol
Chloramphenicol - 15 µg ml-1 in ethanol
Kanamycin - 30 µg ml-1 in dH2O+

Transformation of SCS1 Supercompetent Cells

Protocol is the same for XL-1 Blue competent cells which we also used.
1. Pre-chill Eppendorf tubes on ice (one for each transformation and one for pUC control). Preheat SOC medium to 42C.
2. Thaw the supercompetent (or competent in Xl-1 Blue) cells on ice. When thawed, gently mix and alliquot 53µl of cells into pre-chilled tube.
3. Add 0.9µl of B-mercaptoethanol to each aliquot of cells
4. Swirl contents gently. Incubate on ice for 10 minutes, swirling again every 2 minutes.
5. Add experimental DNA to each aliqout expect for one single tube to which 1µl of pUC control DNA is added.
6. Incubate on ice for 30 minutes. 7. Heat-pulse the tubes in a 42C water bath for 45 seconds. The duration of this is critical for miximal efficiency.
8. Incubate tubes on ice for 2 minutes.
9. Add 0.9ml of preheated SOC medium and icubate the tubes at 37C for 2 hours in shaking incubator. 10. 100µl was plated onto agar plates and 2.5µl of pUC control was plated onto an Amp resistant agar plate.

Transformation of Calcium Chloride competent cells

1. Inoculate one E.coli colony in 5ml of LB medium (liquid) over night.
2. Take 1ml of the overnight culture and add it to 100ml of pre-warm LB liquid in a 1L flask.
3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).
4. Split 100 ml culture into 25 ml aliqouts and incubate on ice for 10min. All the subsequent steps should be carried out at 4°C and cells should be kept on ice as much as possible.
5. Centrifuge for 10min at 3500rpm (4°C).
6. Remove supernatant carefully by pouring and pipetting of the reminder. Place on ice immediately.
7. Resuspend each pellet in 2.5 ml of chilled TSS buffer.
8. Add 100 µl to Eppendorf tubes on ice. Freeze those aliqouts in a dry ice/ethanol bath and sotre at -80°C.

Purification

Protocols