Question on pMiniT 2.0cloning. 1. what is the mechanism of toxic gene or toxicit

Question

Question on pMiniT 2.0cloning.
1. what is the mechanism of toxic gene or toxicity of NEB PCR cloning?
2. If a plasmid self ligated into E Coli, can you expect growth on Ampicilin containing plates? Plates without ampiciliuon? Explainwjy orwhy not.
3. Why vector: insert ratio in a ligation rxn is important?
Thanks !! Question on pMiniT 2.0cloning.
1. what is the mechanism of toxic gene or toxicity of NEB PCR cloning?
2. If a plasmid self ligated into E Coli, can you expect growth on Ampicilin containing plates? Plates without ampiciliuon? Explainwjy orwhy not.
3. Why vector: insert ratio in a ligation rxn is important?
Thanks !!
1. what is the mechanism of toxic gene or toxicity of NEB PCR cloning?
2. If a plasmid self ligated into E Coli, can you expect growth on Ampicilin containing plates? Plates without ampiciliuon? Explainwjy orwhy not.
3. Why vector: insert ratio in a ligation rxn is important?
Thanks !!

Explanation / Answer

1) Ans: If you see the vector map of pMiniT 2.0 (2,588 bp), that two amino acids toxic minigene is located within the MCS ( multiple cloning sites) , which means if your insert gets properly placed within the MCS, by means of restriction digestion and ligation, then it will disrupt the toxic gene expression. That means, your vector, containing insert will only be able to survive. For those vectors without any insert, that toxic gene will be expressed and your transformed colony will not survive. So you are bound to get only truly transformed colonies on your plate. This is how the presence of this toxic gene aids in increasing the efficiency of cloning.

2) Ans: Plasmid is getting self-ligated means, you have a single restriction digestion in your vector and it generates a sticky (Cohesive) end in it, and there is a good chance that it will self-ligate. Now from the pMiniT 2.0 vector point of view, even if it gets self-ligated, and you transformed it into competent E. coli cells and plated in ampicillin, colonies won't survive due to the functioning of that toxic or lethal gene. This vector is designed in such a manner that until or unless this toxic gene sequence is getting disturbed by insertion of your insert, it will kill the E. coli cell.

So with or without ampicillin plates??? It doesn't matter. No colonies will grow.

3) Vector: Insert ratio is very important.

Say for instance, within a 10 microliter of a ligation mixture, there is DNA ligase, your vector, and your insert. Now the vector should be in a vicinity of your insert, and DNA ligase has to be in that space so that it can ligate the insert in the vector. Remember DNA ligase is not going to find your vector/insert and ligate them. So the reaction volume should be adjusted in such a manner that ligase can interact with both insert and vector. That is why it is always recommended that the ligation mixture volume should be between 8-10 microliter.

In such scenario, if the vector size is huge, then no of moles of the vector should be increased, only by then it will increase the individual number of vector and that will automatically increase the probability of interaction of vector and insert. In short, the more number of vector molecule/ insert molecule increases...... the more chance of successful ligation happens. There is a formula, which is used to effectively calculate it:

ng insert to be added = (3)(25 ng vector) (bp of insert/2588 bp of the vector)

Notice here both vector size and concentration has been taken into account.

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