E. coli Electroporation
PROTOCOL DUMP PLEASE FORMAT
Check requirements before starting: 200mL LB, 50 sterile microcentrifuge tubes chilled, 4x 50 mL falcon tubes chilled, 150mL chilled 10% glycerol, 2mL chilled 20% glycerol
Initial step: Overnight cell growth
1) Add 5 ml of LB media into a test tube
2) Add 5 ml /1000 = 5 ul antibiotic (Streptomycin) to the test tube
3) Add E. coli cells (from -80 C freezer) to the test tube by using a tip
4) Put the test tube in the incubator: 37 C and 300 rpm
5) Wait for 12 – 14 hours for cells to grow (The doubling time for E. coli at 37 C is 30 min)
Main step 1: Growing electrocompetent cells
1) Add 100 ml LB media into 2 Erlenmeyer flasks (total 200 ml).
2) Add 100 ml/1000 = 100 ul antibiotic (Streptomycin) into each of the Erlenmeyer flasks.
3) Measure the optical density, OD, of cells grown overnight in the 5 ml test tube.OD of 1for E. coli means10^8 number of cells.
4) We want to have OD of 0.01 at each of Erlenmeyer flasks at the start of the experiment. Therefore, we need to add some overnight grown cells to each Erlenmeyer flasks. The needed amount is calculated as: (100 ml*0.01) / OD = 1/OD.
5) Put two Erlenmeyer flasks in the incubator at28 - 30 deg Cwith 300 rpm (cover the opening of flasks with foil). The reason we grow cells at 30 C is that we want to make them electrocompetent cells. The cell growth at 30 C makes the cell wall structure organized in such a way that it is ready for transfer of DNA into the cells.
7)Be very careful of the cells OD in the Erlenmeyer flasks. The best OD range for the electrocompetent cell is 0.4 to 0.6. Therefore, at least 2 times sampling and measuring cells OD is necessary to monitor the exponential increase in the cells OD. It usually takes 4 - 5 hours for electrocompetent cells growing at 30 C to reach OD of 0.5. The doubling time of E. coli at 30 C is about 40 – 50 min.
Main step 2: Harvesting electrocompetent cells
0) Before starting the experiment , prepare around 50 microcentrifuge tubes (label them) and keep them in -80 C freezer. Prepare an ice-water box too.
1) Turn on the centrifuge and keep its temperature cool at 4 C
2) Take the cells out of incubator when OD reaches 0.4 – 0.6 (Caution needed)
3) Transfer the electrocompetent cells to 4 x 50 mL Falcon tubes while keeping the Falcon tubes in ice water
4) Put Falcon tubes in centrifuge and spin down (8000 RPM, 5 min, 4 C). For the table top centrifuge in our lab, it must be 4500 RPM, 10 min, 4C.
5) Pour out supernatant quickly and be careful of cell pellets.
6)While keeping tubes in ice water, gently resuspend (DO NOT USE VORTEXER) the cell pellets in each Falcon tubes by 25 mL10% glycerol.
7) Transfer the 4 x 25 mL of solution from the 4 x 50 mL Falcon tubes into 2 x 50 mL Falcon tubes (keep them cold)
8) Put Falcon tubes in centrifuge and spin down again (8000 RPM, 5 min, 4 C)
9) Pour out supernatant quickly and be careful of cell pellets.
10)While keeping tubes in ice water, gently resuspend the cell pellets in each Falcon tubes using 25 mL10% glycerol.
11) Transfer the 2 x 25 mL of solution from the 2 x 50 mL Falcon tubes into 1 x 50 mL Falcon tube (keep it cold)
12) Put Falcon tube (along with a balance tube of water) in centrifuge and spin down again (8000 RPM, 5 min, 4 C)
13) Pour out supernatant quickly and be careful of cell pellets.
14)While keeping tubes in ice water, gently resuspend the cell pellets in the Falcon tube in2 ml of 20% glycerol(mix 0.5 ml of 50% glycerol and 1.5 ml of 10% glycerol in order to have 2 ml of 20% glycerol)
15)In the cold room!, aliquot ~55 ul of cells into as many 1.5 mL tubes as possible and as fast as possible.
16) Store them in -80 0C freezer.
* Glycerol protects cells from being killed in -80 C freezer. If only water exists along with the cells in the -80 C, the ice crystals will break the cells wall like a needle and kill them. Therefore, glycerol protect cell against the ice crystals! Moreover, glycerol keeps the structure of electrocompetent cell wall prepared for transformation of DNA. The other role of glycerol is to remove all the salts existing with cells in the supernatant. Salts are very bad for the electroporation step. They make electroporation device arc.
Transformation by Electroporation
Thaw a 50 ul aliquot of electrocompetent cells (EC) on ice. Put an electroporation cuvette on ice.
Add 2-3 ul of concentrated ligation product to electrocompetent cells.
Mix gently by pipetting, but DO NOT generate air bubbles. Transfer to electroporation cuvette.
Wipe metal sides of electroporation cuvette perfectly dry. Place cuvette into electroporator.
Electroporator settings: 2500V for E. coli. Press the ‘Pulse’ button twice. If you hear a popping sound, the conductance of the cells was too high and your cells are dead. This is called ‘Arcing’. The time constant is expected to be between 4.6 and 5.6 for optimal electroporation.
Add 600ul SOB or SOC media to cuvette. Mix by pipetting and transfer to test tube.
Incubate at 37 deg C. The incubation time depends on the antibiotic resistance marker:
Ampicillin:15 minutes
Chloramphenicol:30 minutes
Kanamycin / Streptomycin:45 minutes
Continue protocol @Plating(see below).
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