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Cloning questions - (Jun/21/2006 )

Hello,

This forum's been really helpful to me and I've been reading a lot of your ideas and solutions to various problems. So I decided to post a couple of my questions here:

I am currently trying to clone two things:

1. A 34 base oligo (not phosphorylated when purchased, but I did follow a protocol that was suggested in this forum to phosphorylate it) into a PmeI cut vector (dephosphorylated - blunt end). Somehow I got more colonies on my vector control plate (without the insert) than with the insert. What does this mean? I've had this problem happening with other regular plasmid insert cloning as well sometimes, but just never understood why.

2. I'm also trying to clone a 4 kb insert into an 8 kb vector. Is there a reason to believe that the 4kb might be better off dephosphorylated than 8 kb?

Please let me know if you have any ideas/solutions/suggestions,
Thanks!

-smiles-

1. Did you convert your 34 nuc oligo to a double-stranded DNA before trying to clone it?
Typically, unless you have more than twice as many colonies on the insert plus vector plates as you have on the vector only plates, the chances of finding a colony with an insert is very low.

2. You want to dephosphorylate the fragment that contains the replication origin, designated the vector, in order to prevent it from ligating to itself. Inserts do not usually contain a replication origin and should be phosphorylated.

-tfitzwater-

Thanks so much for your reply. I will dephosphorylate the 4 kb as that's the one that contains the orgin of replication.

Yes, I did anneal both the strands according to the protocol given by IDT.


QUOTE (tfitzwater @ Jun 22 2006, 09:01 AM)
1. Did you convert your 34 nuc oligo to a double-stranded DNA before trying to clone it?
Typically, unless you have more than twice as many colonies on the insert plus vector plates as you have on the vector only plates, the chances of finding a colony with an insert is very low.

2. You want to dephosphorylate the fragment that contains the replication origin, designated the vector, in order to prevent it from ligating to itself. Inserts do not usually contain a replication origin and should be phosphorylated.

-smiles-