In PCR, how does length/composition of primers affect annealing?
In PCR, how does length/composition of primers affect annealing?
Homework Answers
The length of primers and its compositions of its sequence are important parameters which determines the melting temperature as well as annealing temperature of PCR primers. The greater the length of primer higher will be the annealing temperature since it will contain more G+C content. Annealing temperature is in general 5 degree lower than melting temperature of the primers which is given by the following formula.
Tm= 4(G+C)+2(A+T)
Which means annealing temperature also depends on the sequence compositions of A,T,C and G. If the length of primer is unusually long then it affects annealing by delaying it.
1. What are the degenerate primers in the PCR amplification protocol? What do they do? 2....
1. What are the degenerate primers in the PCR amplification protocol? What do they do? 2. Suppose you begin a PCR reaction with 1 piece of double stranded DNA. After 30 cycles of replication, how many pieces of double stranded DNA do you now have? 3. What would be the consequence of having too much DNA in the sample? Would it interfere with the PCR reaction? Why? 4. What happens during the annealing step of the PCR reaction? During the...
How many primers does it take to amplify a single PCR product? Explain
How many primers does it take to amplify a single PCR product? Explain
how to solve this problem about PCR ( no band or faint band )?? 1- PCR...
how to solve this problem about PCR ( no band or faint band )?? 1- PCR product has high GC content . 2-Primers were designed or synthesized incorrectly . 3-Extension time was too short. 4-Incorrect annealing temperature. 5-Denaturation temperature was too low.
Select an annealing temperature based on the Tm of the forward and reverse primers. Briefly justify your selection Sel...
Select an annealing temperature based on the Tm of the forward and reverse primers. Briefly justify your selection Select an extension time based on the expected length of the amplified DNA fragments. Briefly justify your selection
• 4. How many copies of the primers do you add to the PCR? You add...
• 4. How many copies of the primers do you add to the PCR? You add 3 pl. of a 5 M solution of each primer, If you had 100 copies of the template DNA would there be enough primers to support 30 cycles of PCR? Remember the amount of template doubles (in theory) with each cycle. After 1 cycle there will be 2xthe starting amount, after 2 cycles 4 x the starting amount.
3’-TATAAAGACTTACAAATTTGTCCCCATTTTGC-5’ 5’-ATATTTCTGAATGTTTAAACAGGGGTAAAACG-3’ a. Diagram the results you would obtain for 1 and 2 rounds of PCR replication using the primers, 5’-ATGTT-3’ and 3’-CCATT-...
3’-TATAAAGACTTACAAATTTGTCCCCATTTTGC-5’ 5’-ATATTTCTGAATGTTTAAACAGGGGTAAAACG-3’ a. Diagram the results you would obtain for 1 and 2 rounds of PCR replication using the primers, 5’-ATGTT-3’ and 3’-CCATT-5’ and template above. b. The primers in part b were designed to make it easy to illustrate the PCR process, in practice PCR primers are 18-25bp in length. Why do PCR primers that are 5bp fail? c. Are primers used in PCR RNA or DNA? d. Explain how PCR amplifies the gene of interest without amplifying the...
When and how does the RT-PCR test detect the presence of the virus? During PCR. by...
When and how does the RT-PCR test detect the presence of the virus? During PCR. by adding a fluorescent dye and a quencher dye which will be degraded if DNA polymerase finds the viral gene. The sample if positive fluoresces. After PCR, by adding a fluorescent dye which will be activated once the sample goes through a special laser. During PCR, by adding fluorescent primers that are detected through a special laser. After PCR, by adding a quencher dye which...
Problem.1: What does a PCR optimization means? Problem.2: You are working on a gradient PCR to...
Problem.1: What does a PCR optimization means? Problem.2: You are working on a gradient PCR to determine the optimum annealing temperature for your primer, and you get the following result. Problem.5: Your target is to amplify a promoter region of TRF2 gene. You design the following primer pair: Forward: 5'- CAGCGCTGCCTGAAACTC-3: Tm: 58.6°C, length: 18 bp, GC%: 61.11% Reverse: 5'- GTCCTGCCCCTTCACCTT-3' Tm: 59°C, length: 18 bp, GC%: 61.11% Product size: 200 bp -You get the following result: Marker CC CCC...
This PCR step is called annealing. The annealing step follows the denaturation step: it is usually...
This PCR step is called annealing. The annealing step follows the denaturation step: it is usually the lowest temperature in the PCR. The temperature of this step varies with each PCR reaction because each primer has its own sequence and may not be an identical match to the DNA template strand. GC or CG have three hydrogen bonds and AT or TA have two hydrogen bonds. Higher annealing temperatures are more stringent and require a better match between primer and...
How does temperature affect the significance of grain boundary diffusion for changing the composition of a...
How does temperature affect the significance of grain boundary diffusion for changing the composition of a solid?
1. What are the degenerate primers in the PCR amplification protocol? What do they do? 2. Suppose you begin a PCR reaction with 1 piece of double stranded DNA. After 30 cycles of replication, how many pieces of double stranded DNA do you now have? 3. What would be the consequence of having too much DNA in the sample? Would it interfere with the PCR reaction? Why? 4. What happens during the annealing step of the PCR reaction? During the...
• 4. How many copies of the primers do you add to the PCR? You add 3 pl. of a 5 M solution of each primer, If you had 100 copies of the template DNA would there be enough primers to support 30 cycles of PCR? Remember the amount of template doubles (in theory) with each cycle. After 1 cycle there will be 2xthe starting amount, after 2 cycles 4 x the starting amount.
When and how does the RT-PCR test detect the presence of the virus? During PCR. by adding a fluorescent dye and a quencher dye which will be degraded if DNA polymerase finds the viral gene. The sample if positive fluoresces. After PCR, by adding a fluorescent dye which will be activated once the sample goes through a special laser. During PCR, by adding fluorescent primers that are detected through a special laser. After PCR, by adding a quencher dye which...
Problem.1: What does a PCR optimization means? Problem.2: You are working on a gradient PCR to determine the optimum annealing temperature for your primer, and you get the following result. Problem.5: Your target is to amplify a promoter region of TRF2 gene. You design the following primer pair: Forward: 5'- CAGCGCTGCCTGAAACTC-3: Tm: 58.6°C, length: 18 bp, GC%: 61.11% Reverse: 5'- GTCCTGCCCCTTCACCTT-3' Tm: 59°C, length: 18 bp, GC%: 61.11% Product size: 200 bp -You get the following result: Marker CC CCC...
This PCR step is called annealing. The annealing step follows the denaturation step: it is usually the lowest temperature in the PCR. The temperature of this step varies with each PCR reaction because each primer has its own sequence and may not be an identical match to the DNA template strand. GC or CG have three hydrogen bonds and AT or TA have two hydrogen bonds. Higher annealing temperatures are more stringent and require a better match between primer and...
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