Team:Fatih Turkey/ecolitransformation

E.coli TOP10 Transformation                                       

MATERIALS

·         Heat block

·         Incubator with shaker

·         Competent cell

·         Lb broth

·         Lb agar with antibiotic

·         Parafilm

·         Sterile MiliQ dH2O

·         Ice

·         0,5 and 1,5 epp

·         Spreader

 

SOLUTIONS

LB Agar Preparation

- Add 200 mL of dH2O to a graduated cyclindar.

- Transfer dH2O into glass bottle.

- Add 7 gr of LB-agar powder

- Autoclave the bottle.

- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)

- Pour the plates .

LB Broth Preparation

- Add 200 mL of dH2O to a graduated cyclindar.

- Transfer dH2O into glass bottle.

- Add 4 gr of LB powder

- Autoclave the bottle.

- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)

 Transformation

- Aseptic conditions prepared (70% EtOH, Bunsen burner etc.)

- Place 500 uL LB in epp into heat block(42˚C).

- Thaw 50 uL competent cells on ice.

- Add 1 uL plasmid into the competent cell epp and spin for few sec.

- Incubate for 45 min on ice.

- Incubate at 42˚C for 80 sec in heat block.  

 - Incubate for 5 min on ice.

- Complete to 500 uL with pre-heated LB (42˚C).

-.Epp s adhered with tape to horizontal on shaker.

- Incubate at 37 C for 1 h at 240 rpm.

- Spread 125 uL from each tube on agar plates with suitable antibiotic.

- Incubate plates at 37˚C not longer than 12-14 h.