equation, you must add 4 gL of 5 mM MgCl2 to a 200 gL reaction to bring that reaction to 100 gM MgCl2.
Lets look at the following example: MgCl2 will be added to a series of PCR tubes in concentrations of 0.5 mM, 1 mM, 2 mM, 3.5 mM, 5 mM, and 10 mM. The stock solution of MgCl2 you will be given has a concentration of 25 mM. In the spaces provided, calculate the volumes of 25 mM MgCl2 stock solution required for this reaction set to bring each 50 gL reaction to its desired magnesium concentration. Using the 1st approach, you will obtain the result as follows in Table 3.3
Table 3.3
Volume of reaction components for magnesium titration
Notice that the volume of above components row has a variety of values. That means, at this point, that the final volume in your tube will all be different (except Tubes 1M and 2M). So a wise person may ask, «How can all the reagents added be at the same concentration if the final volumes are different?)) And they wouldn’t be.
So let’s take one more thing into consideration and then make the final volumes identical so that the only concentration that is changing is the MgCl2. The final volume of each of the reactions will be made identical by the addition of water.
Remember that the volumes you have entered were all for each tube. That means you have to add 5 pL of PCR buffer to each of 7 tubes. Then add 4 pL of 10 mM dNTP stock to 7 tubes and so on. That is a lot of pipetting and each time you pipet you can introduce some error in the measurement. So we will use a way to minimize the pipetting. Now it’s time to make a Master Mix, which should contain all the reagents needed for the PCR except for the one component being titrated (in this case, magnesium) and the template DNA (which should always be added last to a PCR). The Master Mix will also contain the smallest amount of water any of the 7 tubes will need. Once prepared, an aliquot of the Master Mix will be delivered to each reaction tube. Then, different amounts of MgCl2 will be added to each tube, and additional water will be added to each reaction such that the final volume will be 50 pL. Template DNA will be added as the final step prior to thermal cycling.
Still some part of water needs to be part of a Master Mix so that concentrated salts in the reaction are diluted. This will be the least amount of water every tube will contain. In order to ensure the proper dilutions and volumes additional water might be added. To calculate it it might be necessary to answer two questions:
A. What is the largest volume of MgCl2 being added to any one reaction? 20 qL
B. For the reaction corresponding to this MgCl2 amount, what is the volume of water required to bring that reaction to 50 qL? 9.75 qL
At least this amount of water will be contained in each reaction you will prepare. This amount of water will be used in calculating a Master Mix. To ensure that enough Master Mix is available, calculate the total volumes of each reagent needed assuming 8 reactions must be prepared.
Additional calculations might be necessary in order to calculate the amount of an additional water needed to add to each reaction tube (in addition to the 9.75 qL added to the Master Mix), so that the total volume of MgCl2 plus water is equal to the largest single volume of MgCl2 being added (see Table 3.4).
Table 3.4
Volumes of additional water to add to each reaction in the magnesium salts
No additional water will need to be added to the reaction containing the largest volume of MgCl2. For instance, for Tube 1M the volume of diluted MgCl2 solution to add is 2 qL. The largest single volume of MgCl2 being added is 20 qL. Therefore, 18 qL of additional water needs to be added to tube 1M. Please note that for tube 1M you are adding 2 qL of diluted MgCl2; all other tubes, 2M-7M receive the stock solution (25 Mm MgCl2). Important notes to consider:
– Add reagents to the bottom of the reaction tube, not to its side.
– Add each additional reagent directly into previously-added reagent.
– Do not pipette up and down to mix, as this introduces error. This should only be done when resuspending the cell pellet and not to mix reagents.
– Make sure contents are all settled into the bottom of the tube and not on the side or cap of tube. A quick spin may be needed to bring contents down.
– Keep all the reagents and components on ice.
– Do not forget to label your tubes correct and clear.
– Prepare your Master Mix according to the previous calculations.
– Gently flick the Master Mix tube with your finger to mix the solution. Making sure to use a balance tube, spin MM tube for 5 seconds in a microcentrifuge.
– Template DNA is not added to MM, but individually to each reaction.
– Slowly pipet up and down to mix the reagents after each addition.
– To each tube, add the appropriate combination of MgCl2 and water.
– Do not forget about the positive, negative control tubes.
– Prepare agarose gel while running the reaction.
Agarose is a gelatinous substance derived from a polysaccharide in red algae. When agarose granules are placed in a buffer solution and heated to boiling temperatures, they dissolve and the solution becomes clear. A comb is placed in the casting tray to provide a mold for the gel. The agarose is allowed to cool slightly and is then poured into the casting tray. After it solidifies the gel, in its casting tray, is placed in a buffer chamber connected to a power supply and running buffer is poured into the chamber until the gel is completely submerged. The comb can then be withdrawn to form the wells into which the PCR sample is loaded.
Before switching on the power supply and loading the wells of the gel with sample, one in each, to a small volume of total PCR reaction, it is necessary to add loading dye, which is a colored, viscous liquid containing dyes (making it easy to see) and sucrose, Ficoll, or glycerol (making it dense), mix and then pipet an aliquot of the mixture into the corresponding wells. The samples should be allowed to electrophoresis until the dye front (either yellow or blue, depending on the dye used) is 1 to 2 cm from the bottom of the gel. The gel can then be moved, stained and photographed (often using a Gel Doc system). The DNA is visualised in the gel by addition of ethidium bromide, which, when intercalated into DNA, emits fluorescence under UV-light. Other possibility for visualization, for instance like in a DNA sequencing gel, is an autoradiogram (in case if the molecules to be separated contain radioactivity).
Calculations: for instance, you will need a 2 %, mass/volume agarose gel for electrophoresis of your PCR products. If your agarose gel casting trays holds 50 mL, then how much agarose and buffer would you need? The definition of m/v % in biology is grams (mass) / 100 mL (volume). Therefore, for 2 % agarose, it will be 2 g /100 mL buffer. Step 1: Calculate the mass of agarose needed for 50 mL total volume of agarose solution. Step 2: Calculate the amount of buffer needed to bring the agarose solution to 50 mL. By standard definition, 1 gram of H20 = 1 mL of H20.
The amount of buffer for the 2 % agarose solution will be 49 mL (50 mL – 1 mL {1 gram of agarose}).
Why magnesium chloride is so important? DNA has an overall negative charge because of
