Team:UAAAN/Design
Preparation step 1: 3-7 days before processing
Prepare the agar Plates must be prepared at least 3 days before processing. They must be stored for 2 days at room temperature and then under refrigeration until use. On two days at room temperature, they dry the agar in order to facilitate the uptake of the processing solution.
To prepare the agar:
i. Add 150 ml of distilled water to an Erlenmeye flask.
ii. Add the contents of the LB agar packet.
iii. Shake the flask to dissolve the agar and heat to a boil in the microwave.
iv. Stir again and heat about three times more until the agar dissolves, taking care to cool the flask before stirring, to prevent the hot medium from leaping into the hand.
v. When the agar has dissolved, allow to cool until the flask can be touched without burning (50 ºC).
vi. While the agar cools, label the plates and prepare the antibiotic. Be careful that the agar does not get so cold that it solidifies.
Label Plates
The 10 agar plates should be labelled with an indelible ink marker at the base, near the edge of the plate. Label 2 plates as LB, 8 as LB/[antibiotic].
Add the agar LB on the plates
First, add the agar LB on the 2 plates marked as LB. With one hand open the top of the plate with your fingers holding the bottom with your hand, while with the other hand you add the LB agar. Fill the plate between one third and one half of its capacity ( 12 ml). Cover that plate, and continue with the next one. When all the plates have been filled, allow to cool. Then add the already hydrated antibiotic to the remaining LB agar in the flask. Shake the flask briefly for mixing. Filling the 8 plates labeled as LB/[antibiotic] *-not knowing which vector we will use we do not know which antibiotic resistance gene has inserted in its sequence-according to the technique described above.
Storing the plates
After leaving the plates at room temperature for two days, they can be used or stored in columns and placed in bags. Store the plates in the refrigerator upside down and inside the bags until used.
The LB plates should be seeded to obtain isolated colonies of the bacterium and incubated at 37 ºC for 24-36 hours before processing. The purpose of isolation is to form isolated colonies from a suspension with a high bacterial concentration. Under favorable conditions, one cell multiplies to give millions of genetically identical cells in 24 hours. There are millions of bacteria in a single 1 mm colony.
a) Insert a sterile seeding handle into the bacterial suspension. Insert the handle without tilting the vial. Remove the handle and spread on the plate as shown in the figure below. The extension is done in four quadrants. The first extension is to disperse the cells a bit. Slide the handle from left to right a dozen times in each of the quadrants. In each consecutive quadrant the cells are increasingly diluted, increasing the possibility of obtaining isolated colonies.
b) It is important to use the largest possible plate surface to perform the extension. Rotate the plate approximately 45 degrees (to facilitate hand movement) and start with the second quadrant. Touch the previous quadrant a couple of times and then continue sliding the handle from left to right about 10 times.
c) Turn the plate again and repeat the action.
d) Turn the plate one last time and sow the last quadrant. Repeat steps a-d on the rest of the LB plates. Use the same seeding handle for all plates. When finished with each plate, cover it immediately to avoid contamination.
e) Leave the plates overnight in the inverted position in the incubator at 37 ºC or at room temperature for 2-3 days if no incubator is available. Use for processing within 24-36 hours. Do not refrigerate before use.
f) E. coli produces cream-coloured, round, smooth-edged colonies. Do not use plates contaminated with other colonies.
Preparing the transforming Vector
With a new sterile pipette add 250 µl of the transformation solution in the vial containing the freeze-dried vector. The amount of DNA is so small that it may appear that the vial is empty. If possible, store the rehydrated DNA in the refrigerator (The transformation solution is used because it is sterile and does not contain nucleases. Sterile distilled water can be used instead).
Prepare aliquots
Add 1 ml of the processing solution (Cacl2) and 1 ml of the LB stock to the 2 ml eppendorf tubes. If the LB stock is added 1 day before practice, store under refrigeration. Label the tubes.
After having left the E. coli cells in recovery, observe the plates both transformed and control. Fewer colonies should be observed on the plates containing the vector, since only those that incorporated and expressed the antibiotic resistance gene contained in the vector survive.