Cloning Help! - (Dec/16/2008 )
I have been working on a cloning project with very little success. I am using a vector which I have already put one gene into, and now I am attempting to put another gene in using different single RE site. The insert is small only 300bp, but the vector will end up being about 9kb. I have PCRed out the 300bp insert with RE sites within the oligos. Digest both the insert and the vector; then gel purified both. Since, there is only one RE digest, I treated the vector with SAP to prevent self ligation, then heat inactived. I ligated the two together with T4 DNA polymerase and transformed into DH5a cells. I get a good amount of colonies, and I have a control with just SAP treated vector, which had only one colony. But when I check them with the same RE digest, I don't get my insert back out.
I am running out of ideas...Please Help!
Why don't you try with a different RE(s) to check your product? Actually what you see on the gel after RE of your products? Different bands or a smear.
Well I have several question
T4 DNA polymerase? Is this a spelling mistake? Do you mean T4 DNA ligase.
Are you cutting enough DNA to see 300bp band? Since your plasmid is quite large, you need to cut alot more DNA, as most of that DNA will be the vector backbone, rather than the insert.
Related to this, is there a restriction site within your insert that can allow you to detect the presence of the insert and also give you a larger product band.
Is your T4 DNA ligase in good condition? This enzyme goes bad very easily. Is anyone in the lab suffering problems with ligation?
Did you add enough bp at the ends of the primer? Restriction enzymes require a minimum number of bp around their restriction sites before it can cut efficiently.
T4 DNA polymerase? Is this a spelling mistake? Do you mean T4 DNA ligase.
Are you cutting enough DNA to see 300bp band? Since your plasmid is quite large, you need to cut alot more DNA, as most of that DNA will be the vector backbone, rather than the insert.
Related to this, is there a restriction site within your insert that can allow you to detect the presence of the insert and also give you a larger product band.
Is your T4 DNA ligase in good condition? This enzyme goes bad very easily. Is anyone in the lab suffering problems with ligation?
Did you add enough bp at the ends of the primer? Restriction enzymes require a minimum number of bp around their restriction sites before it can cut efficiently.
Yes, I did make a mistake, I am using T4 DNA Ligase.
I am using 5uL of a standard qiagen mini prep for my digestion, Is that enough? Would it be best to sequence to find out if my insert is in the vector.
There are very few RE sites left and none within my insert. But maybe I can try finding one some place else.
The T4 ligase is about 2 months old and I am the only one using it right now, so I think it is in good shape.
I used a CG pair at the end of my oligos, which I thought was standard. Perhaps I need to add more?
Thanks!
I do not have a lot of options for other RE sites, but that is good idea. As for the gel, I get the vector with out the insert and what I assume is some undigested vector above the ladder.
Thanks!
For many enzymes you certainly need more. What restriction enzymes are you using? Check NEB technical guide to make certain.
So you see the vector piece on the gel! May be because of the size difference you don't see your insert piece on the gel. May be you can use a larger amount of your plasmid in digestion. Also you can run like 2% or higher gel which will allow you to see smaller bands properly.