I have no idea where to start nor what to draw, nor what strategy to take. Pleas

Question

I have no idea where to start nor what to draw, nor what strategy to take. Please tell me what strategies I should take to complete parts a and b of the question and why I should take them as well as why you drew the plasmids and resulting junctions the way that you did. Thank you

1. a) In general, uaing PCR allows you to incorporate different restriction sites on your fragment. Can you design a strategy that would be directional? Draw the resulting plasmid, including the direction of the fragment. (Which restriction endonucleases are directional and which ones are not?)

b) Using the same fragment (from part a) you want to put it into different vector. This vector is identical to pUC18 except the BamH I site is now a Bgl II site (not to be confused with a Bgl I site). Draw the resulting junctions (Vector-fragment). This type of strategy can allow for additional level of section for vector with insert. Before the recombinant plasmids are transformed into bacteria, the mixture is digested with Bgl II. Why does this help?

Explanation / Answer

we always add restriction sites in primers. These restriction sites are same and compatible with the vector in which we want to add our fragment.

3) Directional cloning means that both vector and amplified genes have to be digested with two different compatible enzymes so that our insert fit in one direction in a vector.

steps to be followed (sorry I couldn't upload the image of the plasmid, because there is an error coming right now in image uploading)

1) Design primer with suitable restriction sites. add two restriction enzymes site in primers which are also present in vector.

2) Amplify your target gene with these primers by PCR.

3) digest both your vector and insert with the same enzymes, so that compatible restriction sites are generated in vector and insert.

4) always add restriction enzyme in the forward primer which is present after promoter /ori sequence in the vector and the second enzyme in the reverse primer. this means the enzyme in reverse primer should always come after the forward one otherwise the direction of the insert will get reversed after ligation.

5) then ligate the insert and vector

b) if you want to clone the same insert into another vector remeber that the vector has same restriction enzymes as present in your inert. if you want to clone in different vector with Bgl II site, repeat the same process except add the Bgl II restriction site in one of your primer(taking care of the direction). after ligation of vector and insert the restriction site of Bgl II will be restored.

and it is always recommended to check your DNA before transforming into bacteria this will help you to avoid transforming wrong or contaminated DNA.

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