Primers. (1) PCR primers should be 10-24 nucleotides in length. (2) The GC content should be 40%-60%. (3) The primer should not be self-complementary or complementary to any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation.(4) Melting temperatures of primer pairs should not differ by more than 5°C, so that the GC content and length must be chosen accordingly. (5) The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 25 nucleotides, the approximate melting temperature is calculated with the formula: Tm = 4(G C) 2 (A T). (6) The annealing temperature should be about 5°C lower than the melting temperature.
MgCl2 concentration. Because Mg 2 ions form complexes with dNTPs, primers andDNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg 2 ions result in a low yield of PCR product, and too many will increase the yield of non-specific products. The recommended range of MgCl2 concentration is 1 to 3 mM, under the standard reaction conditions specified.
Taq DNA polymerase. Higher Taq DNA polymerase concentrations than needed may cause synthesis of non-specific products.
dNTPs. The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the reaction mixture is usually 200 μM. These concentrations must be checked as being equal,because inaccuracies will increase the degree of misincorporation.
PCR procedures: equipment
! Micropipettes
! Thermocycler
! Electrophoresis units
! Power supply units
! Photographic equipment
PCR technology in pictures
The following photographs demonstrate how PCR technology is carried out
The PCR mixture is prepared on ice. Safety clothing is not required, but may benefit the reaction unless good sterile techniques are practised.
The PCR reactions are loaded into the thermocycler.
The thermocycler is locked shut and programmed.
Different types of thermocyclers exist: the black one is a tetrad, in which 4 sets of 96 samples can be run simultaneously. The four smaller white ones each have a 96-well capacity.
Depending on the size of the PCR bands produced and the discrimination needed, band visualisation can be accomplished through either a regular, horizontal, agarose gel or a vertical acrylamide sequencing gel (see next slide). Here, the products are being run on an agarose gel.
The acrylamide gel may be run in either an independent unit or an automatic sequencer.The preparation of the sequencing gel, although somewhat complicated to set up, is similar for both cases. Here, the glass is being cleaned and wiped before preparing the gel for an automatic sequencer.
Glass units are being inserted into the frame, which will be fitted into the sequencer.
The glass units are clamped into place, and the entire unit readied for gel to be poured in.
Liquid acrylamide gel is poured into the mould. Because acrylamide is a carcinogen, safety clothing must be worn.
Clamps grip the bottom ends of the glass units to prevent the gel leaking.
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