Team:RAMNOTIREN CALGARY/Protocols

Heat shock

Background:

Competent cells are thawed, put on ice for 30 minuets and then put in a 37 degree incubator for five minuets with the foreign DNA plasmid of choice (this plasmid was pipetted into the tube with the competent cells after they were thawed and prior to putting them in the ice bath). Competent cells are frozen with pores in their membranes, icing them for thirty minuets allows for the plasmid to stick to the cells membrane. When the competent cells are put in a 37-degree incubator the pores of the cell membrane become larger therefor allowing for the plasmid to get in. Putting the competent cells back on ice after heat shocking them causes the pores to become smaller keeping the plasmid inside the cell.

Procedure:
1. Thaw the competent cells that your team made earlier (about 100 μL for each transformation). Remember to keep the cells cool while thawing them.
2. Add between 1 μL-20 μL DNA of choice to the thawed tube of competent cells and mix the DNA by gently pipetting the tube up and down.
3. Once the DNA has been mixed in, leave the tube on ice for thirty minutes
4. After the thirty minutes are up, heat shocks the cells for five minutes in a 37-degree water bath inside a 37-degree incubator. It is most effective if you heat the water to 37 degrees while you have the competent cells on ice.
5. After five minuets, place in an ice bath for five minuets
6. Take off ice bath and add 250 μL SOC broth (used to maximize the transformation efficiency of competent cells).
7. Incubate for thirty to sixty minutes in a shaking incubator, or ninety to one hundred and twenty minuets in a non-shaking incubator.
8. After the incubation period, centrifuge cells to remove all but 100 μL of supernatant. (As 100μL is marked on the 1.5ml tubes it is recommended that you pipette 20μL of the supernatant out until you get down to 100 μL)
9. Re-suspend the cell into solution by vortexing or generally tapping)
10. Place around 50μL of the remaining competent cells onto antibiotic plates (such plate could be one containing LB and chloramphenicol)
11. Let competent cells grow over night on the antibiotic plate at 37 degrees Celsius.
Once you have transformed your DNA into the bacteria, you will need to grow up many colonies of the bacteria. This will allow you to have multiple copies of your DNA to play with. To see how to do this, proceed to the Feeding Bacteria protocol.

Feeding the Bacteria

Background: Feeding bacteria is simply the term used to describe transferring the competent cells (e.coli) containing your DNA of choice into new tubes containing LB and an antibiotic. In this protocol, we used chloramphenicol as the antibiotic. However, you should use the same antibiotic that you had on the plates you put your competent cells on in the last step of the transformation protocol. In transferring the e.coli from one tube to another you keep them happy and healthy. They are happy because they are being given more food, and a home free of waste. You should feed your bacteria every one to two days.

Procedure:
1. In a sterile area, pipette 1000 μL of LB broth into a 1.5 ml tube.
2. Pick one colony of ecoli (grown in the incubator) from the antibiotic plates created in the transformation protocol and place it in the 1.5 ml tube. It is recommended you use a clean pipette tip to pick the colony off of the plate, to get it off the pipette tip swirl the pipette in the LB broth (in the 1.5ml tube) for 20 seconds.
3. Add 10 μL of chloramphenicol (or the antibiotic of your choice) into the 1.5 ml tubes
4. Place the 1.5 ml tube in a non-shaking incubator for 48 hours. This will allow the bacteria to grow and multiply. The tube should be cloudy after 48 hours, telling you your ecoli have grown.
5. Pipette 500 μL of the ecoli (which was grown in the incubator over 48 hours) into a new 1.5 ml tube containing 1000 μL of LB. *Note*-After you have created a 1.5 ml tube of E.coli, you should be feeding or “rehoming” 500 μL of these bacteria every day. This means repeating steps 1-6 everyday.
6. Add 10 μL of chloronphenical (or antibiotic of choice) into the 1.5 ml tube.
7. Repeat steps 1-4 so that you have a new colony of bacteria growing in the incubator.
8. Steps 1-4 should be repeated every two days, while steps 5-8 should be repeated every day until you are completely done all of your assays. In repeating these steps every day or every two days you ensure you have lots of e.coli containing your DNA

Trouble shooting: If you look into your 1.5 ml tubes and notice your bacteria no longer looks cloudy, but streaky, then it means that your bacteria ruptured. They were left in their old tubes too long, and they ran out of food, and too much waste built up for them to survive. You will have to go back a day, and feed the bacteria you rehomed two days ago. If you no longer have these bacteria, or they too have ruptured you will need to create new ones out of the colonies growing on the LB antibiotic plates. Also if for some reason you must leave your bacteria for more then 2 days without being fed, you should put them in the fridge. This will slow down the log cycle and keep the bacteria from multiplying rapidly. In preventing them for multiplying rapidly, you will get less in the tube and they will run out of food slower and produce less waste. This is what had to be done we went 3-4 days without feeding their bacteria.

Once you have grown your DNA in the bacteria, you will want to centrifuge the plasmid out so you can cut and ligate it to use in the sequence you are trying to create.

Agarose Gel Electrophoresis

Reagents and Materials:
1X TAE
Graduated Cylinder
125 mL flask
Agarose
Gel Pouring Tray
Tape
Gel rig
SYBR Safe

Protocol:
Measure out 100mL of buffer
Transfer buffer to 125 mL flask
Weigh out enough agarose to make a 1% gel
Transfer agarose to 125mL flask
Melt agarose in microwave until solution is almost boiling, stirring every 15-20 seconds
Allow agarose to cool (do not let it cool to the point where it is hard)
Add 4 uL of SYBR Safe to the cooling agarose
Assemble the gel pouring apparatus by inserting gate into slots.
Allow gel to cool until flask can be handled comfortably.
Place comb in the gel rig.
Pour agarose into gel tray.
Allow to solidify. While the gel is solidifying prepare the samples. Add your sample and 1 uL 10x Loading Dye, 4 uL of DNA and 5 uL of water
Pour 1X TAE over gel so that gel is covered by a 3-5mm buffer
Load samples into lane (Don't forget to load a 1kb+ ladder into one of the lanes)
Hook electrodes to gel apparatus.
Run the apparatus at 100V for 30 - 45 minutes (make sure to watch that the dye does not run off the gel)
Record the results.