Molecular biology people and students help me

[ManBearPig]

Im preparing for the final tomorrow, and although i have a general idea of what the answers are to these (study checklist) questions, im not positive. Here it goes:

Why would a researcher bother to use directional cloning rather than bi-directional cloning?

What potential technical problem does the MCS (multiple cutting site) circumvent?

Unlike bi-directional cloning, in directional cloning cut plasmids which do not anneal with an insert fragment fail to circularize. Is that good or bad? Why?

Why is the term insertional inactivation very descriptive, and make good sense as a name?

How does the presence of an antibiotic resistance marker on the plasmid help the researcher?

What is the point of using x-Gal in the culture medium in the final screen?

 

[Cerpin Taxt]

42

 

[PhoKingGuy]

Man i'm bored

Why would a researcher bother to use directional cloning rather than bi-directional cloning?

To ensure a gene is inserted in the correct direction in your vector of interest.

What potential technical problem does the MCS (multiple cutting site) circumvent?

Telling you where your inserts can be added in via different restriction enzymes

Unlike bi-directional cloning, in directional cloning cut plasmids which do not anneal with an insert fragment fail to circularize. Is that good or bad? Why?

good, because it increases the yield of your transformation reaction. Additional positive screens can be carried out via other methods (hybridization of probes, etc)

Why is the term insertional inactivation very descriptive, and make good sense as a name?

Ive never heard that before

How does the presence of an antibiotic resistance marker on the plasmid help the researcher?

By being able to screen for the presence of the plasmid or not. If it grows on the antibiotic medium, it will have taken up and expressed the plasmid

What is the point of using x-Gal in the culture medium in the final screen?

X-gal screens for the presence of Beta Galactocidase enzyme expression

I hope you're just checking, this is pretty basic stuff you're asking for having a final tomorrow

 

[TheVrolok]

I thought about answering these. Then I realized that google would be easier for the both of us

 

[Ophir]

It's been a while, but I'll take a stab at it:

Originally posted by: Kazaam
Im preparing for the final tomorrow, and although i have a general idea of what the answers are to these (study checklist) questions, im not positive. Here it goes:

Why would a researcher bother to use directional cloning rather than bi-directional cloning?

You would use directional cloning when it matters what orientation the DNA is, e.g. when you want the organism to produce a particular peptide. If it is bi-directional, and you inserted your fragment after the promoter, the RNA polymerase would transcribe two different RNAs.

What potential technical problem does the MCS (multiple cutting site) circumvent?

No idea.

Unlike bi-directional cloning, in directional cloning cut plasmids which do not anneal with an insert fragment fail to circularize. Is that good or bad? Why?

I would think it is good since a linear fragment is not viable and would be easily identifiable by gel electrophoresis - you'd see a linear plasmid band. It would be an easy way to tell if the plasmid took the DNA or not.[/quote]

Why is the term insertional inactivation very descriptive, and make good sense as a name?

No idea.

How does the presence of an antibiotic resistance marker on the plasmid help the researcher?

It helps because the cloned bacteria will be able to grow on antibiotic (ampicillin) containing media, bacteria that did not take the DNA will not grow. This is another way to identify if the plasmid took the DNA as well as a great way to isolate such bacteria.

What is the point of using x-Gal in the culture medium in the final screen?

No idea.

Sorry if I'm totally off, just trying to point you in the right direction. All my work now is on the protein side - someone else deals with growing that crap and getting me the right protein.

 

[jagec]

Shooting from the hip here, but...

a)Directional cloning ensures that the ORF is inserted in the correct direction for expression (i.e. in-line with the promoter, if it's backwards it won't express). Also, it prevent the vector from self-ligating (creating vector w/o your insert), since the RE sites will be different. If you do blunt-end cloning or a single RE digest, you have to screen for colonies that inserted "correctly"; if you do directional cloning they should all be correct. Given that everyone has a sequencer sitting in their back pocket, though, I always sequence to ensure there were no screwups. But I only sequence a couple of clones, since they should all be correct.

b)Well, for one, if your insert contains a RE site that you do not wish to cut, you can use different enzyme(s) to cut it out if you stuck it in an MCS-containing plasmid. It also facilitates directional cloning, future cloning on the same plasmid (some inserts will destroy a RE site...this way you can still cut using that enzyme, or use a different enyzme to ligate a new insert), and of course helps if you run out of the enzyme you wanted to use and don't want to wait for a shipment. Also, if you're doing a multiple digest, some enzymes don't play well in the same buffer (although you can almost always find a buffer that works, and increase the digest time if needed)
It's just more flexible than having to tie yourself to single enzymes.

c)Good! Bacteria won't replicate non-circularized plasmids, so the only clones that will grow up will contain plasmids with your insert in place.

d)First you have a correctly functioning gene, and then you insert some DNA in the middle of it, and it no longer functions. Sounds pretty apt to me! Usually you do this with a gene with has a very obvious and measurable effect on your bacteria, like GFP (they will no longer glow green under UV if the gene is inactivated), or a resistance marker (they'll die when exposed to antibiotics)

e)If you grow transformants on media containing antibiotic, the only bacteria that will grow are those which contain your plasmid and therefore are resistant. It also means you can be fairly lazy about contamination. This is especially useful if you're sick and sneezing all over the place.

f)Blue-white colony screening. A plasmid containing a functional LacZ gene with an internal MCS is used to transform bacteria following a ligation. If your ligation worked, the gene will be inactivated, and the bacteria won't metabolize the X-gal. If your ligation did not work, the enzyme will work, metabolize X-gal, and turn blue.

 

[ManBearPig]

you guys rock, thank you so much! I know it's basic...i understand it, but our teacher is horrible and he writes the book, so i like to get a second opinion/source when i can.

appreciate it everyone!