Primer 1 lul Primer 2 luI Template DNA 3. Experimental steps 1.Taq PCr MasterMix Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C Deoxynucleoside triphosphates(dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase Divalent cations, generally Mg2+ is used 2. Denaturation step This step is the first regular cycling event and consists of heating the reaction to 94-98 C for 20-30 seconds. It causes DNA melting of the DNa template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA 3. Annealing step: T he reaction temperature is lowered to 50-65 C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. The polymerase binds to the primer-template hybrid and begins DNA synthesis The temperature at this step depends on the dNa polymerase used commonly a temperature of 72 C is used with Tag polymerase. At this step the dna polymerase synthesizes a new DNA strand complementary to the dna template strand by adding dnTPs that are complementary to the template in 5to 3 direction 5. Final elongation 70-74 C for 5-15 minutes after the last cycle to ensure that any remaining single-stranded DNA is fully extende 6. Final hold. I is step at 4-15 C for an indefinite time may be employed for short-term storage of the reaction To get special gene from genomic DNA by PCR. 6. PCR lid100℃ Initialization step 94 C8min 35 Cycles: Denaturation step 94 C30s Annealing step 55℃C30s Extension step72℃50s Final elongation 72℃10min Final hold NOTES 1. Primers(short DNA fragments)containing sequences complementary to the target region along with a DNa polymerase (after which the method is named)are key com 2. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an• Primer 1 1ul • Primer 2 1ul • Template DNA 2ul 3.Experimental steps 1.Taq PCR MasterMix Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C. Deoxynucleoside triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations; generally Mg2+ is used 2.Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA. 3.Annealing step: T he reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 4.Extension/elongation step: The temperature at this step depends on the DNA polymerase used; commonly a temperature of 72 °C is used with Taq polymerase. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction. 5.Final elongation: 70–74 °C for 5–15 minutes after the last cycle to ensure that any remaining single-stranded DNA is fully extended. 6.Final hold: T his step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.To get special gene from genomic DNA byPCR. 6.PCR lid 100℃ Initialization step ：94℃8min 35 Cycles： Denaturation step 94℃30s Annealing step 55℃30s Extension step 72℃50s Final elongation ： 72℃ 10min Final hold 4 ℃ NOTES 1.Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. 2.Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an