We have had multiple successes cloning 20-30 Kb fragments in plant transformation vectors that themselves are >10 Kb, so it is definitely possible. We also use electroporation with home-made electrocompetent cells as we believe that we get higher transformation frequencies with large plasmids. I have a few suggestions for you:

1. As Boris Wawrik suggests, you should make sure your competent cells are actually competent. Use freshly diluted pUC plasmid and use about 100 pg per transformation to calculate how competent your cells are.

2. Prior to transformation, clean up your ligation mixture. Typically this is not needed, but if you are having trouble, it is an easy fix. We use the old-fashioned phenol/CHCl3 extraction followed by CHCl3, and ethanol precipitation. Sometimes we use carrier (about 20 µg of glycogen) during the precipitation. Dissolve your pellet in TE.

3. If you are indeed cloning a PCR product, you should NOT phosphatase your vector UNLESS your PCR primers were synthesized with 5' phosphates.

4. Prior to the ligation step, we clean up the insert and vector (phenol/CHCl3, CHCl3, ethanol precipitate) and determine the DNA concentrations using a fluorometer or running a sample on a gel. The more accurate the concentrations, the more likely the ligation will work.

5. I have never observed ligase "going bad", but if you suspect your ligase is not working, you should test it using digested plasmid.

Good luck!

You might want to try electroporating the competent cells in different (higher) voltages as the plasmids you want to construct is gonna be big ones.

Use some pUC19 to QC your competent cells. 7kb is not a large fragment. Have you checked for homology between your insert and the vector ? What vector ? Are you using the right antibiotic ? I'm assuming you are trying to clone a PCR product. Are you using high fidelity TAQ ? Run your ligation on a gel to see if your ligase worked. Also, clone with two sites, not just one. Try different ratios of DNA : vector.

Have you considered using recombineering to do this? This technique uses the lambda bacteriophage recombination genes (lambdaRED system, see Datsenko and Warner, PNAS 2000). You add at least 50 bp of homologous DNA to the ends of your cloning insert and then using cells that are induced for expression of the lambdaRED genes, you electroporate both your target vector and the insert (with homologous DNA on the end) into the cells at the same time.

Have you tried Gibson assembly cloning?

We have had multiple successes cloning 20-30 Kb fragments in plant transformation vectors that themselves are >10 Kb, so it is definitely possible. We also use electroporation with home-made electrocompetent cells as we believe that we get higher transformation frequencies with large plasmids. I have a few suggestions for you:

1. As Boris Wawrik suggests, you should make sure your competent cells are actually competent. Use freshly diluted pUC plasmid and use about 100 pg per transformation to calculate how competent your cells are.

2. Prior to transformation, clean up your ligation mixture. Typically this is not needed, but if you are having trouble, it is an easy fix. We use the old-fashioned phenol/CHCl3 extraction followed by CHCl3, and ethanol precipitation. Sometimes we use carrier (about 20 µg of glycogen) during the precipitation. Dissolve your pellet in TE.

3. If you are indeed cloning a PCR product, you should NOT phosphatase your vector UNLESS your PCR primers were synthesized with 5' phosphates.

4. Prior to the ligation step, we clean up the insert and vector (phenol/CHCl3, CHCl3, ethanol precipitate) and determine the DNA concentrations using a fluorometer or running a sample on a gel. The more accurate the concentrations, the more likely the ligation will work.

5. I have never observed ligase "going bad", but if you suspect your ligase is not working, you should test it using digested plasmid.

Good luck!

Thank all for helpful suggestion.

To Boris, my two insert size is 6650 and 6498 respectively (as mentioned above) and the two recipient vector is about 12.4 kb and 7.2 kb respectively. So, the final ligated product will be around 19 kb and 15 kb respectively. I have isolated my insert from another vector, pUC19 with single enzyme digestion (PacI) and trying clone the insert into the plant transformation factor which pPIPRA56o using the same enzyme. I am pretty sure that I am using right antibiotic with right concentration.Also my ligase is working as I have make many clone at the same with this ligase.

As sad already above, recombineering and assembly might be the better choice, instead of classical cloning. You can use recombineering for example from Gene Bridges. Good luck!

I have very good results with electroporation using dH10B (invitrogen) in combination the electroligase kit from NEB. The great thing is ,you don't need to clean up your ligase reaction before electroporation. And yes, always use the puc control to asses the transformation efficiency. The electroshox were not so efficient in my hands.

Also cloning large constructs, they sometime are not as stable as

Sorry i am not clear, have you confirmed you recombinant construct by sequencing or digestion? First you confirm the recombinant construct, for which you can design one primer from vector and another from insert and carry out PCR from ligated mixed product. If you are sure that ligation is successful than only go for transformation. Generally 50 mirogram/ml is used for kanamycin selection. You can try transformation with know vector in host.