Team:Tec-Monterrey/projectprotocols

1.Measure 60 ml of TBE (.5 X) with the probe.
2.Take a beaker and place 60 ml of TBE.
3.Place the filter paper on the scale.
4.Weigh 600mg of agarose gel on the scale.
5.Mix 600mg of agarose gel in TBE and heat it in the microwave for time intervals of 10 seconds.
6.Place the electrophoresis comb in the tray electrophoresis.
7.Pour the solution into the electrophoresis tray, making sure the level does not reach beyond the teeth of the comb. Wait until it solidifies.
8.Remove the electrophoresis comb and place the tray electrophoresis in the electrophoresis chamber.
9.Pour TBE by the sides of the electrophoresis chamber until the level of TBE exceeds the gel.
10.Mix the DNA samples, molecular weight ladder and DNA supercoil with 12μl of loading buffer and SYBR Green 4μl.
11.Load the wells with the samples made in the previous step.
12.Connect the electrophoresis chamber to the power supply at a voltage between 60V and 70V and run it for 1.5 hours.
13.Use transilluminator to see results.

1.Perform the necessary calculations using the calculator developed by the Tec-Monterrey 2010 team. (click here)
2.Add the required amounts of buffer, plasmid DNA, nuclease free water and BSA (keep reagents in ice except the sample DNA).
3.Pour the required amount of enzyme at the end.
4.Incubate for 1 hour at 37 ° C.

1.Take 1ml of transformed cells in a 1.5ml eppendorf tube.
2.Centrifuge for 1min at 10,000 rpm.
3.Discard the supernatant. Repeat step "1" 5 times.
4.Take 250μl of Cell Resuspension Solution to resuspend the pellet.
5.Take 250μl of Cell Lysis Solution. Immerse it 4 times and incubate for 4min.
6.Take 10μl of Alkaline Protease Solution and incubate for 4min.
7.Take 350μl of Neutralization Solution. Immerse it 4 times.
8.Centrifuge at 14,000 rpm for 10min.
9.Place the minicolumn in a collection tube.
10.Remove the supernatant with a point and place it in the minicolumn.
11.Centrifuge for 1min at 14,000 rpm.
12.Remove the liquid from the collection tube and replace the minicolumn.
13.Take 750μl of Column Wash Solution and place it in the minicolumn.
14.Centrifuge for 1min at 14,000 rpm.
15.Remove the liquid from the collection tube and replace the minicolumn.
16.Take 250μl of "Column Wash Solution" and place it in the minicolumn.
17.Centrifuge for 2 min at 14.000 rpm.
18.Remove the liquid from the collection tube. Take the minicolumn and place it in a 1.5ml microcentrifuge tube.
19.Take 100µl of nuclease free water and pour it in the minicolumn.
20.Centrifuge for 1min at 14,000 rpm.
21.Store at -20 ° C.

1.Cut the band of interest and weigh.
2.1μl of Mem Bind Solution will be placed for each mg of gel.
3.Heat the gel with a water bath at 50 ° C to dissolve the gel.
4.Incubate at 25 ° C for 1min.
5.Place the minicolumn with its collection tube.
6.Pour the liquid incubated in the minicolumn.
7.Centrifuge at 14,000 rpm for 2min.
8.Decant the solution of the collection tube and replace the minicolumn.
9.Add 750μl of Mem Wash Solution to the minicolumn.
10.Centrifuge at 14,000 rpm for 2min.
11.Decant the solution of the collection tube and replace the column.
12.Add 500μl of Mem Wash Solution to the minicolumn.
13.Centrifuge at 14,000 rpm for 2min.
14.Decant the solution of the collection tube and replace the minicolumn.
15.Centrifuge 1 min.
16.Take the mini-column and placed in a 1.5ml eppendorf tube.
17.Pour 35μl nuclease-free water in the minicolumn.
18.Centrifuge at 14,000 rpm for 1min.
19.Store at -20 ° C.

1.Cells should have an optical density of .4 (always keep tubes on ice).
2.Centrifuge at 5,000 rpm, 2 ° C for 5min.
3.Decant supernatant.
4.Add 1 ml of CaCl2 to resuspend the pellet.
5.Add 20ml CaCl2.
6.Centrifuge 5,000 rpm, 2 ° C for 5min.
7.Decant supernatant.
8.Add 1 ml of CaCl2 to resuspend the pellet.
9.Add 20ml CaCl2.
10.Centrifuge 5,000 rpm, 2 ° C for 5min.
11.Decant supernatant.
12.Add 500μl of glycerol + CaCl2.
13.Store at -80 ° C.

1.You need to have calcium competent cells.
2.Take 50μl of cells and mixed with 2μl of BioBrick resuspended.
3.Place on ice for 15min.
4.Place in microwave on level 2 for 1min.
5.Place on ice for 1min.
6.Add 200μl of LB.
7.Incubate for 1 hour at 37°C with stirring.
8.Cultivate in a dish 50µl of bacteria and 150µl in another dish.

1.Take a pre-inoculum of virgin cells and pour in 15-20ml of LB agar without antibiotic.
2.Make an Overnight (15-16hrs).

1.Streak Escherichia coli DH5α cells onto LB-agar plate with no antibiotics and incubate at 37°C overnight.
2.Pick one colony and place it in a 50 mL tube with 20 mL LB medium. Incubate overnight on a shaker at 37°C and 350 rpm.
3.Add 250 ml of LB medium to a flask and add the overnight culture until an OD600 of 0.1 is reached.
4.Place the flask on a shaker at 37°C, 350 rpm until an OD600 between 0.4-0.6 is reached.
5.Transfer the diluted culture to 50 mL tubes.
6.After this step, the cells must be kept at 4°C at all times. Place the cells on ice for 15 minutes.
7.Cool the centrifuge to 4°C.
8.Centrifuge the tubes for 10 min at 8000g at 4°C.
9.Remove supernatant and gently resuspend pellets with 10 mL cold sterile water by pipetting. Add the rest of the water to a total volume of 50 mL.
10.Centrifuge a second time for 10 min at 8000g at 4°C.
11.Remove supernatant and gently resuspend pellets with 10 mL cold sterile water by pipetting. Add the rest of the water to a total volume of 50 mL.
12.Centrifuge a third time for 10 min at 8000g rpm at 4°C.
13.Remove supernatant and gently resuspend pellets with the remaining water (if it’s too little, add some more).
14.Calculate and add glycerol so that the final concentration is 10-15 %.
15.Resuspend the cells and aliquot 50 μL per 0.2 mL tube (tubes on ice) and store at -80°C.

1.Chill electroporation cuvettes, DNA samples and tubes on ice.
2.Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice.
3.Turn on electroporator and set voltage to 2.5 kV.
4.Dial a micropipette to 1 or 2μL of DNA sample.
5.Dial a micropipette to 50μL of electrocompetent cells.
6.Dial a micropipette to 1000μL and pipet in SOC. Place micropipette on counter such that tip doesn't touch anything.
7.Pipet 1-2μL of DNA sample and place inside the cuvette.
8.Pipet 50μL of electrocompetent cells inside the cuvette ensuring they mix with the DNA sample. Do not pipet up and down.
9.Place cuvette back on ice to ensure it remains cold.
10.Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.
11.Wipe off excess moisture from outside of cuvette.
12.Place in chamber of electroporator so that the cuvette sits between electrodes.
13.Pulse the cells with a shock by pressing button on electroporator.
14.Remove cuvette from the chamber and immediately add SOC.
15.Transfer cuvette to 37°C incubator and shake at 350 rpm to promote aeration. Incubate for 1 hr.
16.Plate 100 μL transformation onto LB-agar plate supplemented with appropriate antibiotic.
17.Incubate plate overnight at 37°C until colonies appear.

1.Take an Eppendorf tube and add the required amounts of reagents.
2.Mix gently and centrifuge so that the contents of the tube to the bottom.
3.Incubate at room temperature for 5 min.
4.Use 2μl of the ligation reaction to transform 100µl of competent cells.

1.In a microfuge tube mix in the following order:
a)10X Amplification Buffer (5µl)
b)20mM Solution of four dNTPs pH=8.0 (1µl)
c)20µM forward primer (2.5µl)
d)20µM reverse primer (2.5µl)
e)1-5 units/µl thermostable DNA polymerase
f)H2O (28-33µl)
g)Template DNA (5-10µl)
h)Total Volume (50µl)
2.Program the thermocycler at the necessary conditions for denaturation, annealing and polymerization.

This is the recipe to prepare an Acrylamide Gel (10%):
1. Place the resolving solution on the glass.
2. Add 1ml of water, why? Water help’s the gel to no have irregularities on the surface.
3. Let polymerize for 30 minutes.
4. Dry the water on the glass with filter paper.
5. Add the stacking solution.
6. Add the comb carefully, avoid the formation of bubbles.
7. Let stand for 1 hour and a half.
8. WATCH OUT acrylamide is neurotoxic, DO NOT swallow.
Note: Unlike other protocols, PSA cannot be prepared instantly. After you prepare it, you can refrigerate it at 4°C. When using PSA, It is highly recommended to avoid sudden temperature changes. You can use a cooler to prevent the temperature changes.
Note: PSA and TEMED are polymerizing agents (MUST NOT inhaled). Once both compounds are added, must be added nimbly to the solution in the glass, because these compounds may start polymerizing once it´s added. You need abilities, if you have a Thelma in your team leave her this work.

Soluble and insoluble protein extraction from bacterial cell culture & Preparation of samples for SDS-PAGE

6 ml of transformed cell culture is harvested by centrifugation at 10 – 12,000 x g for 1 min.
The supernatant is removed and the pellet is dried.
For each 1 g of cell pellet, 20 ml of xTractor Buffer, 40 µl of DNAse 200 µl of 100X lysozyme solution is added.
The suspension is incubated during 10 min at room temperature.
The crude lysate is centrifugated at 10 – 12000 x g for 20 min and the supernatant is called as soluble fraction and the pellet as insouble fraction.
The insoluble fraction is sonicated with water during about 5 sec.
50 µl of protein fraction is mixed with 50 µl of 2x sample buffer with 2-ME and heat 10 min at 95 °C.