Some Important Techniques and Procedures that I have Used

At the very beginning of my research project, I have learned the procedure of the preparation of the cast an agarose gel. In a short and precise way, the procedure is as follows:

-          measure 50 mL of 1x TAE buffer and pour it in an Erlenmeyer flask;

-          measure and add, into the flask, 0.4 g of agarose (i.e. a white, fine powder);

-          mix the solution by gentle swirling which should give a very cloudy/milky solution;   

-          put the solution in a microwave oven for 60-80 seconds to get a clear solution;

-          let the solution cool down for about 10 minutes;

-          add 50 μL of Ethidium bromide solution* into the warm solution;

*Be careful with this- it's a mutagen and suspected carcinogen, so don't let it spill or get on your hands. 

-     pour the whole solution into the plastic gel chamber and place the plastic comb, with sufficient number of teeth to the number of samples we are testing, to create indentations;

-     wait until the gel would solidify within around 15 minutes;

-     take the comb out and move/place the gel on to the gel apparatus**;

**This is made of hard plastic and consists of an electrophoresis chamber with two wells on the side, a removable gel tray, a comb with as many teeth as you have samples/ladders, and a lid connected to power cords. Place the gel tray in the electrophoresis chamber so that the rubber gaskets on the tray engage the sides of the chamber. You may have to wet the gaskets with water so that they slide in. 

-          fill the gel and the whole apparatus with 1x TAE buffer to cover the sample in depth;

-          then carefully pipette a load of sample into each indentation, recording which sample corresponds with which lane;

-          into one lane alongside those containing your samples, load 5 µL of  DNA ladder, which contains DNA fragments, and 1 µL of 10x concentrated gel loading solution;

-          cover the electrophoresis chamber with the plastic cover, and plug in the two power chords so that the black, negative one is next to the indentations that you have loaded the samples into, and the red, positive one is opposite them;

-          start running the gel for 30 minutes which is indicated by bubbles released; and, finally,

-          remove the gel, place it under a UV lamp, and photograph.

To transform DH5α E. coli with the plasmid:

-          heat shock method was used to transform DH5α cells with the plasmid sample for cloning purposes. 

-          100 μL of CaCl₂ competent cells were divided into two tubes, with one serving as a control tube for the transformation. 

-          3 μL of the plasmid was added to one tube. 

-          Then both samples were heat shocked at 42°C for 45 seconds, 400 μL of NB media added, and incubated with shaking at 37°C for one hour. 

-          The samples were placed on pp LB Broth (100 μL /mL) plates with a sterile stick and incubated over night at 37°C. 

-          Colonies of the transformed cells were then grown in the pp LB Broth medium with shaking for 2 nights at 37°C. 

To isolate the plasmid from the DH5α cells:

-          Two tubes were filled with 1.2 mL of the cell culture.

-          They were centrifuged at 12,000g for 30 seconds at 4°C and the supernatant was removed. 

-          They were mixed by inversion several times and stored on ice. 

-          150 μL of a solution was added to each tube. 

-          The samples were vortexed gently for 10 seconds and incubated on ice for 5 minutes.  They were centrifuged again at 12,000g for 5 minutes at 4°C. 

-          400 μL of each supernatant was transferred to a new tube. 

-          2 volumes (800 μL) of room temperature ethanol was added, the samples mixed by vortexing, and allowed to stand at room temperature for two minutes. 

-          The centrifugation was repeated at 12,000g for 5 minutes at 4°C.  The supernatant was removed and the samples were dried using a speed vac. 

-          Both samples were redissolved in 50 μL TE (pH 8) with RNase (20 μL /mL) by briefly vortexing.

-          The two samples were run on an agarose gel and examined under UV light for analysis of the plasmid bands.

To prepare the buffers for DNA extraction:

-          30 mL of P1 buffer = 0.1818 g Tris base + 0.1116 g Na₂EDTA.2H₂O + 24 mL distilled water + 3 mg RNase A.

-          30 mL of QBT buffer = 1.3149 g NaCl + 0.3138 g MOPS + 24 mL distilled water + 4.5 mL pure isopropanol + 0.45 mL Triton x-100 solution + 0.405 distilled water.

-          200 mL of QC buffer = 11.688 g NaCl + 2.092 g MOPS + 160 mL distilled water + 30 mL pure isopropanol.

-          50 mL of QF buffer = 3.6525 g NaCl + 0.303 g Tris base + 40 mL distilled water + 7.5 mL pure isopropanol.

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