blunt ended cloning problem - (Oct/04/2005 )

Hi everyone

I have a rather complicated question.

I want to clone the 5' untranslated region of a mRNA upstream of the luciferase gene (and in frame) into pGL3 (Promega).

Its complicated at there are only two restriction sites available in the plasmid, Hindiii and NcoI. Conveniently, Nco is at the start codon (ccATGg).

I have designed primers to PCR the utr from genomic DNA, and i want to blunt end the fragment into the HindIII and NcoI sites. (cant just cut plasmid with nco as this would create another ATG upstream of my utr).

Also, my mRNA doesnt have a consensus Kozak sequence and hence doesn't have an NcoI site at its start codon, other wise i could have created primers with an NcoI site and used sticky end cloning. and there's a hindIII site in the PCR product so i cant do that at the 3' end either.

I've had numerous attempts by doing plasmid double digests, filled in the 5'overhangs with klenow then Ciap'd and ligated to cleaned up PCR product treated with klenow to remove 3' A, but no colonies. I've just read i should not ciap plasmid or should kinase the PCR product, so that might explain it.

Has anyone got any other advice - what about using T4 DNA pol instead?

Thanks very much
getting increasingly
frustrated!!

---

Well as you say this is pretty complicated First thing you should know Blunt end cloning is anyway 10 to 50 times more difficult to achieve than cohesive ends ligation. I had very good results in the past using a 5x ligation buffer from Gibco BRL I remember that it was containig PEG which some how help to get the partner of the ligation close to each other.

I had also at that time co precipitate the vector and insert together to make sure they are well mixed (not sure that it's really helpful).

I don't get clearly what you want to achieve you want to clone upstream fromyour luciferas gene and in frame (why that if it's UTR ?) an UTR of a genomic DNA you want to express it or what usage I don't get it !

For sure if you treat your vector with CIAP you have to do it the hard way meaning
30mn 37°C with 0.1units of diluted CIAP
followed by
15mn at 56°C to open up extremities
then repeat the same two steps after adding newly CIAP (same amount)

hope it helps you a bit

pesji

---

I agree with Pesji about the type of buffer ... PEG containing buffers work really well with blunt end ligations.

---

Could TA cloning of your product into an intermediate vector help?

Barring that, I overcame a similar problem (all sites available in my vector also existed in my PCR product) using the BD In-Fusion Kit (I used the dry-down kit).

Basically, you design a set of primers that incorporate on their 5' end 15 bp homologous with your linearized vector. Mix your PCR product with your vector in the BD In-Fusion reagents, incubate for 30 minutes, and transform competent cells.

This was a God-send to me, as for this particular experiment, I could come up with no other way to clone the product I needed in frame (it was a his-tag fusion I was making). I even got my BD rep to give me a sample of the kit, and it worked the first time (I didn't get a huge number of colonies, but all I checked were correct by sequencing), thus I got out of a sticky situation without spending anything...

Protocol-at-a-glance guide here, full manual here...

Hope this helps!

---

Hi everyone,

Thanks for your suggestions, i'm going to check out the clontech kit. I cant use TA cloning as i would have no way of removing exactly the bases i need. Just to clarify, i want to clone the UTR upstream so that the start codon of the original gene is replaced by the start codon of luciferase. I'm then going to see what effect the UTR has on translation in vitro.

Thanks again
frustrated

---

Good luck!

---

Homebrew,
just looking at the in-fusion kit now.
It says if your cloning vector is larger that 5kb (as mine is) you don't need to incubate it in their reagent, just mix pcr product and DNA, then transform.

what do you think -does the method just rely on the homology of the primers and the plasmid which is used as a template for new DNa synthesis in the cells.

---

Hi, I don't fully understand what you are trying to do but I might be able to offer some suggestions

1. I don't know why you are worried about frame in the 5' UTR. Frame only matters in the open reading frame (ATG to stop).
2. I have built a ton of constructs by blunting with T4 polymerase. It works better than Klenow. One key is to make sure that you do the reaction in the supplied buffer. You will probably have to use a cleanup column but they are very easy to use, cheap and quick. I like qiagen's columns. They advertise them as PCR cleanup columns but the can clean any enzymatic reaction
3. I think you might be able to do the cloning with sticky ends. Are you familiar with engineering restriction sites into your primers? Its easy to do. I build almost all of my constructs this way. You can pick whatever sites you want and put them on the ends of the primers. For example, lets say you want to engineer an EcoRI site into your forward primer. Your primer would look like this:

NNNNGAATTCXXXXXXXXXXXXXXXXXXX


The N's represent any bases, you need to add about 4 bases to the end of the PCR product because most restriction enzymes can't cut right at the end of a strand of DNA. GAATTC is the EcoRI site. The X's are the nucleotides that match the sequence you want to amplify. You can engeineer any sequence you want on the end of a primer including the kozac sequence.

---