Questions on PCR 1. You ran a gel to analyze the results of a PCR and you see fo
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Questions on PCR 1. You ran a gel to analyze the results of a PCR and you see four bands in the lane when you expect only one. What are the two most common variations could you use to try to reduce the products to one band. (Sometimes no amount of effort gets you the one band that you want, but you always try.)? 2. You ran your PCR with the following machine parameters: 94°C for 1min, 55°C for 30sec, 72 C for Imin. You expected a PCR product of 3Kb but saw nothing on in the lane on the agarose gel. What should you change to get the product you want? 3. If you started with 10 copies of a DNA fragment and you ran a PCR for 5 cycles, how many copies of the DNA would you have at the end of the run? 4. The forward primer for your PCR has this sequence: 5-TACGGTTAACGCGTATGCTG-3'. What is the Tm for this primer? 5. An agarose gel shows that your PCR of a chipmunk gene succeeded in producing a product of 2Kb which is what you expected. You clone and sequence the PCR DNA product. The result of a computer analysis show that the DNA that was sequenced was human and not chipmunk. What went wrong? What could you do to look out for this problem?Explanation / Answer
Answer) While running a gel electrophoresis of PCR products, we can use both positive and negative controls. and their potential outcome is described below;
Negative control - this is a PCR reaction set, which had all the PCR components like other PCR reactions except it doesn't have the DNA added to the mixture. this way, we can identify any sort of DNA contamination in the chemicals. because this reaction must not give any amplification. but, if this reaction shows any amplification band on gel, it means that either of the chemical is contaminated with non-desired DNA.
positive control - this is not a PCR reaction set, but it contains the initial extracted DNA. this initially extracted DNA is loaded on gel as a positive control to make sure that the desired DNA is amplified in PCR reactions. this band will be of similar size with the desired amplified bands.
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