Cloning a part of a small DNA insert into a plasmid by cutting out the insert!

[tman_ap]

Question:

You are going to clone a part of this small DNA insert into a plasmid by cutting out the insert with SexAI restriction enzyme, opening up the plasmid with SexAI, and then ligating the two pieces together. Given the sequances of the insert and part of the plasmid (below), show where the SexAI sites are in both sequances and then write the sequence of the part of the plasmid including the insert once the pieces have been ligated. Indicate the insert, the plasmid, and the SexAI sites on the new sequance.

 

Insert: 5' CGATACCTGGTGATTGTAAGCCACC... 3'

 

Plasmind: 5' GTAATGCCATTCACCTGGTTTAGGC... 3'

 

 

What I know:

I know that the SexAI enzyme cuts at:

5'ACCWGGT 3'

3'TGGWCCA 5'

However, W is replaced with T 5-3' and with A from 3-5' here!!!

5'ACCTGGT 5'

3'TGGACCA 3'

 

It cuts as fallowing:

 

Insert: 5' CGATA || CCTGGTGATTGTAAGCCA || CCTGGTTAACG 3'

 

 

Plasmind: 5' GTAATGCCATTCA || CCTGGTTTAGGCATGATCT

 

NOW??

 

I am stuck here and I don't know what else to do. Please Help!!!

[ecoli]

are you purposefully cutting your insert? If so why?

 

After you cut your plasmid, you can just ligate the insert and the plasmid; in a specific ratio (which depends on the sizes of the plasmid and insert).

 

In other words... I don't understand what the problem is.

[tman_ap]

It's a genetics assignment.

 

So, after I cut off the site from the SexAI, I just attach the rest of the sequence (after the ligase) together?

 

If yes... do I then attach the plasmid and the insert together with each other to form huge sequence?

[ecoli]

It's a genetics assignment.

 

So, after I cut off the site from the SexAI, I just attach the rest of the sequence (after the ligase) together?

 

If yes... do I then attach the plasmid and the insert together with each other to form huge sequence?

 

No, you digest the plasmid, put in the insert and then ligase.

[tman_ap]

So, I am suppose to cut the plasmid and the insert with the SexAI enyme.. then join the insert I cut parts together and insert THAT insert into the area I cut the plasmid and then connect the other parts of the plasmid with the insert in between?

 

If not, please explain more. I am so confused at this moment!

[CharonY]

The thing you may have missed is that the endonuclease creates sticky ends.

So you digest the plasmid and the vector carrying the insert with SexAI individually.

Then you mix the digested fragments. Due to the overhangs the insert can then attach to the digested plasmid and then get ligated.