Sure, larger products are trickier but that size should work.

Do you restriction digest your PCR product or insert it straight as it is? What polymerase do you use in you PCR? I am asking since this affects how the ends of your product look (Taq or Pfu?). What restriction enzyme do you use to open up your vectors with? Just to make sure you have compatible ends.

Also, if you run your PCR product on a gel and cut it out on a UV table, make sure you work VERY fast. Prolonged UV-exposure of your product will decrease you efficiency in getting a functional clone.

I am using Phusion polymerase. I try to insert two products. One via BamHI and EcoRV and the other one by SalI and BamHI. There should be no compatible ends. I open the vector with the same enzymes, I am digesting my PCR products with.I tried to ligate the PCR products in pcDNA3.1(+) and pIRES2-eGFP.I am working with a UV table and I allways try to work as fast as possible.

Sometime ago I had similar problems. UV table turned out to be a problem. If you have prominent band of your product after PCR reaction then just use column purification (without gel extraction). Simply avoid UV table.

5 kb should not be a problem for pcDNA vector. pIRES was always trickier to work with in our hand, especially, epression-wise. I don't think UV is a main problem, although you need minimize exposure - one way to do it is to decrease intensity of light and keep you gel on Saran wrap. For best rate of transformation gel-purify your insert and vector, and use highly competent bacterial cells (usually commercial one works best, although they are expensive). Also, to help troubleshooting, you can try to subclone a smaller "known good" insert, say 0.5kb, in parallel.

We used gateway vectors for up to 12000 bp fragment cloning. It works fine.

It depends on answers to following questions:

What is the host cell type: mammalian or bacterial or both?

what Tag you want to use?

Which promoter?

Which antibiotic resistance gene to selection?

Hi Andreas For any cloning vectors theorically smaller inserts will be cloned more efficiently than longer inserts. In our lab, we use both TOPO vectors and pGEM- T vectors. We have been able to clone up to 3 Kb insert with TOPO but we have been able to clone more efficiently with pGEM-T systems. We never been succesfull to clone more than 4 Kb insert with T- vectors as far as I remember. If you want to try T-vectors. I suggest you to use pGEM-T Easy with some optimization as time and temperature of ligation, insert-to-vector ratio, time and temperature precision. I warning you to pay attention to the quality of your DNA, competency of your bacteria. Beside, it would be interesting to compare ectrocompetent cells vs chemical competent cells ! I wish you the best.

In my experience the ration of insert to vector is the most important factor (provided you have clean DNA and compatible restriction sites).

Look at this link which provides a good example for calculating the ratio.

Cheers,

https://www.mcdb.ucla.edu/Research/Arispe/Protocols/Vector_Insert_Ratio.pdf

Hmm... so, does other clonings work well? Just trying to rule out the ligation kit/bacteria etc. Other things I can think of are:

- do you have enough extra nucleotides outside your restriction sites in your PCR? Many restriction enzymes work very poorly if they cleave on the very end of a PCR product. Usually 4-6 nucleotides extra outside the actual restriction recognition sequence is enough.

- you are using the right antibiotics in the plates/cultures? If using ampicillin, make sure you have fresh plates to avoid satellite colony formation (or use carbenicillin plates which work for amp-resistance, but no satellite colonies form).

1. Design two primer pairs one for your amplicon without restriction overhang sites(primer pair A) and one with (primer pair B) (add 3 to 6 nucleotide more apart from your RE sites) (BamHI and XhoI always works from me for pcDNA 3.1+)

2. PCR Amplify your amplicon with "primer pair A" using PFU or Phusion polymerase.

3. Take 1/10th your PCR product from step 2 and amplify with 'primer pair B' with fewer cycles than your previous PCR (say 15 to 20 cycles).

4. Make two equal half of your PCR product and run it on agarose in two separate lane without ethidium bromide (etbr).

5. After the run,cut the gel in between two lanes, stain one half with etbr and visualize the band under UV, mark the region of the band(do not cut).

6. Combine the two halfs(stained and unstained) under normal light source and cut corresponding region of the unstained half.

7. Elute the DNA and do restriction digestion

8. Clean the restriction product with PCR clean

9. Do the ligation and cloning. It should work

I hope this helps

The UV table as said above will certainly be the cause of the problem. Moreso, the storage of your pcr fragment is critical, especially if it was eluted in water. exonucleases could have cleaved off the ends. Try using fresh pcr product out of the uv table, and just use purelink PCR cleanup kit with Buffer B3 to clean ur digestion product rather then gel

I have worked with TOPO vectors. If you are intending to perform your cloning into a TOPO vector, then for your case, TOPO-XL (fro EXTRA LARGE) cloning kit would be a suitable vector

We use pGEM-T Easy vector and TOP-10 competent cells for cloning PCR-products for up 10kb size fragments.

I am currently working with TOPO vector to clon a 4.8 kb fragment and it works. Nevertheless, I had to screen many colonies to find those ones with the whole insert.