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Carrier-mediated facilitated diffusion proteins may become saturated when: - There is a maximum size of particle that can be transported by facilitated diffusion. - The concentration gradient has to be of a certain steepness for facilitated diffusion to begin. - A cell can only make sufficient ATP up to a certain maximum rate to fuel facilitated diffusion. - Electrical charges equalize and facilitated diffusion shuts down. - All the protein transporters are occupied and the rate of facilitated diffusion cannot be increased further.

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6.- Your friend Sylvester wishes to subclone a 1.7 kb DNA fragment into the pCR 2.1 TOPO plasmid vector. His goal is to amplify the desired fragment by PCR. At the end of the PCR cycling reaction, he immediately adds 4 μL of PCR reaction (the maximum amount of DNA allowable) to the ligation mix. He did the transformation and plated it onto solid media containing the right amount of X-gal and ampicillin. He picked a single white colony, inoculated it into liquid LB media with ampicillin, and grew it up overnight. The next day, he purified the plasmid DNA from the colony and sent it for sequencing. He prepared two tubes of DNA, one sample of plasmid will be sequenced using the M13-20 primer to sequence the bottom strand. The second DNA sample will be sequenced with the M13 Reverse primer to sequence the upper strand. Sylvester got the sequence back, and the chromatograms were very good. When he BLASTed the sequences using NCBI, he realized that he had cloned a PCR product that was only 800 bp. He was upset because he felt that since he'd used blue-white selection and chose a white colony, he should have isolated the fragment he was interested in. He is confused and does not understand his results. (i) Why was Sylvester able to conclude with confidence that the PCR product he submitted for sequencing was only 800 bp? (10 pts). (ii) Explain why an 800 bp sequence was ligated into the pCR 2.1 TOPO vector although his aim was to amplify a larger PCR product (5 pts). (iii) Give him some recommendations that would help him select the right clones for sequencing in the future (15 pts).
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Question Text
Carrier-mediated facilitated diffusion proteins may become saturated when: - There is a maximum size of particle that can be transported by facilitated diffusion. - The concentration gradient has to be of a certain steepness for facilitated diffusion to begin. - A cell can only make sufficient ATP up to a certain maximum rate to fuel facilitated diffusion. - Electrical charges equalize and facilitated diffusion shuts down. - All the protein transporters are occupied and the rate of facilitated diffusion cannot be increased further.
TopicAll topics
SubjectBiology
ClassClass 12