rDNA applications question BSc lvl

the attached file is a question I do not know how to study for, would be very helpful if one of u could link me with a video or page to study. Would be very nice if I could get the answer.

first post on this site, do forgive me if I am not following the common etiquette.

A sincere and whole hearted thank you,

adam

Original post by adams786

the attached file is a question I do not know how to study for, would be very helpful if one of u could link me with a video or page to study. Would be very nice if I could get the answer.

first post on this site, do forgive me if I am not following the common etiquette.

A sincere and whole hearted thank you,

adam

Hey, I may be completely wrong with these answers, as I am only a lowly first year medical student, and I haven't done this sort of thing, but I can try :P

But anyway, for a) I'd say that 100% of BamHI sites can be cut with MboI. BamHI specifically needs to cut between 2 G's, but Mbol just cuts between a G and any other base, which could include G.
for b) again, I would say 100%?
for c) It would if the bases adjacent to the MboI cleavage site were G's
d) yes, as the only way you could ligate the fragments is if the restriction site was the same as that targeted by BamHI.
e) Obviously the 2 fragments can't ligate because you'd be trying to ligate a blunt end with a sticky end, and likewise, the ends cannot be cut by BamHI because they can't ligate in the first place.

I may be completely wrong, I am tired, but I answered those using common sense, so you shouldn't need revision material, unless I am completely wrong! But like I said, I don't do this sort of thing at uni, so maybe someone with more experience could correct me if I am incorrect

Reply 2

adams786

OP

Original post by AortaStudyMore

Hey, I may be completely wrong with these answers, as I am only a lowly first year medical student, and I haven't done this sort of thing, but I can try :P

But anyway, for a) I'd say that 100% of BamHI sites can be cut with MboI. BamHI specifically needs to cut between 2 G's, but Mbol just cuts between a G and any other base, which could include G.
for b) again, I would say 100%?
for c) It would if the bases adjacent to the MboI cleavage site were G's
d) yes, as the only way you could ligate the fragments is if the restriction site was the same as that targeted by BamHI.
e) Obviously the 2 fragments can't ligate because you'd be trying to ligate a blunt end with a sticky end, and likewise, the ends cannot be cut by BamHI because they can't ligate in the first place.

I may be completely wrong, I am tired, but I answered those using common sense, so you shouldn't need revision material, unless I am completely wrong! But like I said, I don't do this sort of thing at uni, so maybe someone with more experience could correct me if I am incorrect

aaah thank you by the way! lets see if any one could confirm
p.s. i dont ave any common sense

Reply 3

Muz_333

1

A) 100%

B) 100%

C)
5' GATCC 3' Yes.
3' CTAGG 5'

D) Not always. It would only be cut if MboI was cut after a G base. Any other base would not complete the BamHi recognition site.

E(c))
5' GAGATCC 3' Nope.
3' CT--------G 5'

E(d) Sticky ends will not ligate with a blunt end (without chewing back the ends first, an additional enzymatic step out of scope of this question).

Full disclosure, I'm drunk right now. However, I'm a PhD student that deals with this daily, so I'm 99% sure I've got this correct.

Edit: I'm not sure how to study for this. Perhaps sign up for Benchling, a free online to for plasmid map creation and sequence alignments. it also show restriction digest sites so perhaps just play about with it.
To begin with I had to draw out all the steps on pen and paper before my PI told me about tools to do it in a less painstaking manner.