Restriction enzyme digest - (Jan/27/2009 )

I am doing a subcloning experiment and have some questions about the digest.

My insert is in EcoRI and XhoI. My vector has EcoRI and XhoI sites but they are only 5 base pair apart. The vector also has SalI sites. The NEB catalog shows that XhoI and SalI have compatible ends.

So my question is can I digest the insert with EcoRI and XhoI and the vector with EcoRI and SalI and then ligate.

Thanks.

xyz74 on Jan 28 2009, 08:13 AM said:

The idea sounds OK.
Keep looking through the NEB website. There will be pages that tell you about how efficient different enzymes are close to the ends. You might need to one, clean up, then do the other.

yes, you certain can.

However please note there have been problem with SalI ligations. More frequently than expected and for reasons unknown, people encounter problems when using SalI in their ligation strategy. So be advised of potential ligation difficulties.

perneseblue on Jan 28 2009, 02:43 PM said:

Yes, definitely go to the SalI page in NEB.com for their FAQs.

As already pointed out, you may have problems with salI with some plasmids and some it works like a charm.

Hi everyone, I just wanna ask how long do you guys usually leave your enzyme digestion? Is 3 hours enough?
Has anyone tried making mutated libraries using error-prone PCR and doing an enzyme digestion on the PCR products so they can be cloned into vectors?

Thanks in advance.

jiajia1987 on Feb 3 2009, 11:28 PM said:

3 is good enough .

regarding the mutated library ... i think it's quite a nasty business. as you do not know where the error will occur and it might occur more than once albeit rarer.

However, it definitely depends on some factors such as how long the PCR product is , how error-prone is the polymerase so on so forth.

I would leave it for an hour. It should be plenty of time.

You could try site directed mutagenesis instead of error prone PCR.

I did something similar in that I random mutagenized the gene by doing a gene specific pcr and then ligating into the vector. You would need to use primers that are specific to the vector your library is currently in since there are thousands of different open reading frames. Not sure why you would do this since libraries are used to isolate single genes or screen for specific genes. You then mutate the single cDNA you isolated in whatever experiment or screen you preformed (what I did). But, it can be done. Do the pcr, dpn1 digest the original template library, digest with your restriction enzymes, pcr purify or precipitate (note that you will products of many different sizes so gel purification won't work), ligate. Problem is you will most likely loose a number of cDNAs. Why not attempt "round the world" pcr where you amplify the entire vector using taq? This way each cDNA gets amplified and hopefully mutated. You just treat the pcr with dpn1, transform and amplify the mutated library.

We did this to try to identify binding mutants. We had no clue where to place a site-directed mutagenesis so we went on a fishing-expedition and just randomly mutated the gene. Did two hybrid screens and identifed clones that interacted the way we wanted. Isolated the cDNA and sent for sequencing to identify where the mutation occured. Mine had two mutations which we then tested individually by doing site-directed mutations. Managed to identify a single residue critical for interaction and function in the protein of interest.

hanming86 on Feb 4 2009, 08:28 PM said:

Yes, i think it is a nasty business trying to produce a mutated library.I have been having lots of problems getting a mutated library and I am supposed to use error-prone PCR. Just a few days ago, I finally optimized my PCR in such a way that I got the PCR products I want. FYI, I have not been getting the PCR products that I want so it was a pleasant surprise.

Treated it with Dpn1 and did a reamplification after that. I did a double digestion on my PCR products. When I ran it on a gel, the band was quite faint. This is seriously annoying because I need to use the digested PCR products for my ligation. Funny thing is, though the band for my digested PCR products are very faint, I can get high concentrations of the digested PCR products. The highest I got was over 200ng/ul, which was a surprise. What usually happens when I run a gel after enzyme digestion is that the band can become so faint that it can barely be seen unless I overexpose the gel. I am not too sure why this happens.