E-coli RFP Transform

This is an extremely basic & rough protocol to undertake a simple GFP transformation. As the facilities at the London Biohackspace favour the use of electroporation to yield competent cells and do not have -80C storage this protocol also covers the method to prepare cells for electroporation.

- Please feel free to improve on this version, it is what I used for my first successful transform -

Materials

Here is a basic materials list per my transform. You can of course adjust the quantities and materials, this is how I did it.

You will need:

• 6000RPM Centrifuge
• Incubator Shaker
• Electroporator
• Suitable Ecoli Strain
• Suitable GFP Plasmid
• Chloramphenicol
• LB Medium
• Agarose
• Brewing Glucose

Also, you need some basic laboratory supplies:

• Pipette (50μL to 5mL) & tips
• Petri Dishes
• Conical Tubes (15mL and 50mL)
• Lidded Laboratory Bottles (200mL x 4)
• Deionized Water
• Ice Bath

Priming The Ecoli

In this section we will sanitize the input ingredients and culture the Ecoli to be transformed.

1. In a 200mL bottle prepare 200mL Deionized Water with 5g LB Medium [Mix A]
2. [Mix A] Dissolve the LB and then autoclave, remembering to keep the lid slightly untightened to allow the pressure to balance
3. Remove [Mix A] from the autoclave and cool to a comfortably touchable temperature (~40C)
4. Beside a burner or filtered air supply add 4mL of [Mix A] into 2x 15mL tubes [Mix B]. Keep [Mix A] for later
5. Using a sterile sample stick, holding the Ecoli source petri dish upside down, scrape a sample of bacteria. Introduce this into [Mix B] (do not double dip)
6. Place [Mix B] in the incubator and wait until they are cloudy (~8hrs)
7. Once [Mix B] is cloudy reintroduce both 15mL tubes into [Mix A] and place [Mix A] into the incubator until cloudy [~5hrs]

While [Mix A] is incubating you can prepare the media (plural)

• One [Mix A] is cloudy remove from the incubator and put into the ice bath for 30min

Media Preparation

In this section we will (again) sanitize the input ingredients, prepare petri dishes with our Chloramphenicol, and prepare the post shock LB media.

1. Per steps 1-2 above, prepare two 200mL bottles with deionized water and 5g LB. [Mix C] includes 4g Agarose, [Mix D] includes 2g Glucose
2. Prepare a 200mL bottle of deionized water
3. Autoclave [Mix C], [Mix D], and the water. Remove from autoclave when finished
4. Set [Mix D] aside for later, place the water in the ice bath
5. Once [Mix C] has cooled to a comfortable touch but without beginning to solidify, beside a filtered air supply add 50μL Chloramphenicol and mix
6. Pour [Mix C] onto the smaller-diameter side of 6-8 petri dishes, cover and leave to solidify. Labeling these is a good idea

Making Competent Cells

In this section we will repeatedly filter our Ecoli with deionized water

1. Picking up [Mix A] from the ice bath in Priming The Ecoli, separate into 4x 50mL tubes
2. Centrifuge these at 6000RPM for 10min then promptly return to the ice bath
3. Drain the liquid
4. Pipette 5mL of the ice-cold autoclaved water into each of the drained tubes and resuspend the Ecoli
5. Return the tubes to the ice bath for 5min before centrifuging
6. Repeat steps 3-5 one or two more times
7. Following the final centrifuge drain the liquid and resuspend in 500μL of cold deionized water

Electroporation and Plate

In this section we will separate each tube into shock cuvettes, add the plasmid, electroporate, then resuscitate the newly transformed cells (Note, if you haven't sterilized the cuvettes please see the section at the bottom)

1. Take the 500μL tubes of suspended Ecoli and separate into 50μL cuvettes
2. Pipette 1μL of GFP plasmid solution into each & mix (Note: You should avoid innoculating one or two as negative controls)
3. Place the cuvettes into the ice bath (carefully) and leave for 10min
4. Once by one:
   1. Remove the cuvette
   2. Dry it thoroughly
   3. Place it in the electroporator set to 2000V τ=5ms and run
   4. Remove and add 400μL of [Mix D] to the cuvette
   5. Place the cuvette upright into the incubator
5. After the cuvettes have been incubating for 30-45min remove them and apply each sample to two of the petri dishes you prepared earlier
6. Place the petri dishes in the incubator and leave for 24(+)hrs

• To check the success of your transform place the dishes under the appropreate UV light source. The transformed samples should fluoresce

Appendix Notes

Sterilizing Cuvettes

1. Place the cuvettes in 70% isopropyl alcohol and leave for 10 min
2. Remove and drain them of solution, placing them upside down on paper towel to evaporate dry