You are given three primer sets by which to perform PCR with an unidentified bac

Question

You are given three primer sets by which to perform PCR with an unidentified bacterium pure culture sample.

Primer set A: 1492R & F27

Primer set B: F343 & 534R

Primer set C: R1492 & F519

Identify the expected length of each product for each of the given primer sets

Primer set A: 1492R & F27 - Product length = _____________

Primer set B: F343 & 534R - Product length = _____________

Primer set C: R1492 & F519 - Product length = _____________

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Which primer set gives you the largest amplicon?

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What does a large amplicon mean for bacterial identification through the molecular method of identification (this means that you will need to think about the entire process from PCR through BLAST'ing, what does a larger amplicon mean, versus a shorter amplicon, in terms of your ultimate ability to identify an unknown bacterium)?

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You are given three primer sets by which to perform PCR with an unidentified bacterium pure culture sample. Primer set A: 1492R& F27 Primer set B: F343&534R Primer set C: R1492 & F519 Identify the expected length of each product for each of the given primer sets (% pt each) Primer set A: 1492R & F27-Product length Primer set B: F343 & 534R-Product length- Primer set C: R1492 & F519-Product length Which primer set gives you largest amplicon ( pti? What does a larger amplicon mean for bacterial identification through the molecular method of identification (this means that you will need to think about the entire process from PCR through BLAST'ing, what does a larger amplicon mean, versus a shorter amplicon, in terms of your ultimate ability to identify an unknown bacterium)? (2 point) After performing PCR with each of the primer sets and the same pure culture sample, you ask your research colieague to load and run an agarose gel electrophoresis in order to double check that PCR was successful. Unfortunately, your colleague forgets to write down which lane was loaded with which PCR primer set reaction and therefore you are left to identify which band is the result of which primer set on your own. Given the following electrophoresis picture label the Ladder (L), three bands as they correspond to primer set A, B and C and label where the positive & negative electrodes are (½ pt each, 3 pts total).

Explanation / Answer

In this question three primers and their location at different positions were given. In order to find out the PCR size we will minus the value of reverse primer to forward primer and that will give the length of the amplicon.

For set A= 1492-27= 1465 bp long PCR product.

Set B= 534-343=191 bp

Set C= 1492-519=973 bp

In this prime 1465 bp will give the longest product.

Here the longest product will help to find the bacterial molecular identification because it sequence/s the largest chunk of the DNA and that will help us to find out the conserved residues/sequence of the gene. Moreover, largest sequence will provide the wide range of the restriction digestion pattern that will help in designing the restriction map of the bacteria.

As mentioned in previously that gene set A given the largest product hence firs lane that is near to ladder and PCR product near to the well represent the gene product of the set A primers

Set B primers represent the product of 191 bp and that move the fastest, hence, it will present at the bottom and similarly the set C primers give the PCR of 973 bp it give fall between the above mentioned two products. So the lane order is A, B and C.

Here DNA is always negatively charged due to presence of the negatively charged phosphate group. It will move towards the positive electrode. Here upper side, where the loading wells are present, must be a negatively chaged site and bottom electrode is positively charged site.

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