PCR – diagnostics. Aizhan Zhussupova
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 the negatively charged oxygen molecules along the two sugar phosphate chains of the double helix. Since both chains are negatively charged, they have a natural tendency to repel each other. In fact, if DNA is placed in water free of any ions, the two strands of DNA are very likely to come apart. Positive ions such as Na+ and Mg++ (found in sodium chloride and magnesium chloride), however, can interact with the negatively charged DNA strands to mask the forces of repulsion. The higher the salt concentration, the more likely DNA will remain double-stranded.
In addition, at higher salt concentrations, two strands of DNA can be made to anneal to each other even if there is no perfect complementarily between them. Under conditions of very high salt concentrations, the double helix structure for some DNA segments can be quite stable, so much so that an even higher temperature is required to denature it. But the magnesium salts is not the only important part of the PCR (see Fig. 3.1).
Figure 3.1. Comics on PCR: Please… just send the Taq. No more little tubes of magnesium!
Popular blog from Promega Connections (http://promega.word- press.com) top ten tips for successful PCR:
– Modify reaction buffer composition to adjust pH and salt concentration.
– Titrate the amount of DNA polymerase.
– Add PCR enhancers such as BSA, betaine, DMSO, nonionic detergents, formamide or (NH4)2S04.
– Switch to hot-start PCR.
– Optimize cycle number and parameters like denaturation and extension times.
– Choose PCR primer sequences wisely.
– Determine optimal DNA template quantity.
– Clean up your DNA template to remove PCR inhibitors.
– Determine the optimal annealing temperature of your PCR primer pair.
And if you want to, you can even build a custom PCR protocol using their iOS and Android device apps at: http://worldwide.promega. com/resources/mobile-apps/.
Saying it shortly, the hot start PCR is a technique that reduces non-specific amplification and offers the convenience of PCR set up at room temperature, avoiding a non-specific amplification of DNA by inactivating the Taq polymerase at lower temperature (see Fig. 3.2).
Figurе 3.2. HоtStart-IT®mеthоdhttp://www.affymetrix.com/catalog/131145/USB/HotStart-IT+Taq+DNA+Polymerase
Polymerases used in Hot Start PCR are unreactive at ambient temperatures. Polymerase activity can be inhibited at these temperatures through different mechanisms, including antibody interaction, chemical modification and aptamer technology. At permissive reaction temperatures reached during PCR