How do I make XL1 Blue competent cells?

How do I make XL1 Blue competent cells?

1. Autoclave 100 ml LB- broth in a 1000 ml flask.
2. Sterilize and pre-chill the 50-mL conical tubes.
3. Prepare 30 ml of ice cold MgCl2-CaCl2 solution (80mM MgCl2, 20 mM CaCl2)
4. Prepare ice cold 0.1 CaCl2.
5. Pre-warm the LB plates.

What are Electrocompetent cells?

Electrocompetent cells are made competent using an electrical pulse from an electroporator to create temporary pores (poration) in the cell membrane of either prokaryotic or eukaryotic cells.

How do cells prepare for electroporation?

For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.

1. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
2. Place recovery SOC in 37°C water bath.
3. Pre-warm LB-antibiotic plates at 37°C.
4. Thaw cells on ice for 10 min or use freshly made cells.

How do competent cells work?

Typically, researchers use chemical (and heat shock) or electroporation means to transform, although other methods exist. The process of making competent cells introduces pores into the cell membrane which allow they to uptake extracellular DNA more readily. Once these competency methods are complete, the E.

How long are Electrocompetent cells good for?

As long as cells do not got through thaw and unthaw cycles, they should be able to work for 3 years. I have kept cells for longer.

How do you make Electrocompetent E coli?

Electrocompetent E. coli

1. Grow an overnight culture of each strain in LB medium.
2. Prepare 10 ml of fresh LB medium in a 50 ml flask for each strain.
3. Inoculate with 100 μl of the overnight, stationary-phase culture.
4. Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.

How do Electrocompetent cells transform?

Procedure

1. Prechill the cuvettes on ice.
2. Thaw the electrocompetent E.
3. Add the DNA solution (ca.
4. Dry the cuvette with a tisue to avoid a bypass.
5. Push the two buttons until the beeping!
6. Add immediately 1 mL SOB (no antibiotics!) and mix by gentle pipetting.
7. Incubate the cuvette 30-45 minutes at 37°C.

How do Electrocompetent cells take up DNA?

Electrocompetent cells work using the electroporation process. Electrical pulses created pores that allows genetic material to permeate the bacterial membrane. Invitrogen offers a variety of electrocompetent E. coli cells to reliably clone your DNA with high efficiency.

How do you make an Electrocompetent cell?

Why do we use competent cells?

Competent cells, designed to take up foreign DNA from the surrounding at a higher efficiency, are routinely used in molecular cloning to propagate and maintain cloned DNA in plasmids.

Can you Electroporate chemically competent cells?

Electrocompetent cells are prepared to cope with electrotransformation and chimiocompetent cells are made to be transformed via heat shock. If you run electroporation with chemically competent cells, you will get a very nice electric arcing because of the calcium chloride present in cell sample.

How do you make Electrocompetent cells?