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31 Jan 2019

Explain how the Bcl1 –BamH1 fragment can be cloned into a single BamH1 site

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Related questions

you want to compare the 16s rRNA DNA sequence you have cloned tothe ones your classmates have cloned. You are able to purify the16s ribosomal fragment and do restriction digest on it. You use arestriction enzyme that recognizes a 4 base pair site rather than a6 base pair site. Why?

Hi please help with this its very important for my grade worth 25 points of my grade:

The AT-vector containing the rac PCR sequences AND the expression vector DNA to be used for cloning are digested with XhoI and EcoR1. The fragments were then mixed and joined together before transformation.

(a) (4 points) – Why were two different restriction enzymes used?

(b) (4 points) – If the cloning had been done with only a single restriction enzyme (both PCR primers contained the same restriction enzyme site and the vector was digested with only that one enzyme [for example EcoR1]), what additional step would have to be done to ensure that the correct clone was obtained?

(c) If the vector DNA to be used for cloning is digested with BamH1 and the TA-vetor/rac DNA used for cloning is digested with Sau3A, would using these two enzymes as described work for a cloning experiment? Explain

(d) (4 points) - What is the name of the enzyme used to join the ends of the target and vector DNA molecules together?

(e) (4 points) - What would happen during the ligation reaction if the restriction enzyme was not heat inactivated before the ligation reaction?

(f) (4 points) - Why was it necessary to treat the vector with phosphatase?

Briefly explain how blue-white screening works. Include the genes and chemicals needed for this to work properly. Which output would you expect when you have cloned a DNA fragment into a plasmid for blue-white screening? Explain all of the controls necessary to ensure that a cloning experiment with blue-white screening was successful

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