pUC19 based plasmids can easily be used for up to 10kb DNA inserts as long as there are no unusual features in the DNA that may cause their own issues. As the size goes up, the copy number of plasmid molecules/cell will reduce significantly.

I agree with Prof. Premont that the size of insert wouldn't have any effect on the expression in pEGFPC1. We did express KIF13b motor protein (more than 5Kb in size) and ryanodine receptor 1 (~15Kb in size) cloned into pEGFC1 as GFP-fusion proteins in HEK293 cells without any problem.

Dear all,

Thank you for your replay. It was very helpful and informative. Can the limit be in the use of bacteria for transformation you think? Im now using One Shot® TOP10 Chemically Competent from ThermoFisher for my transformation, which has worked very well before. Can you suggest a more suitable bacteria for transforming large constructs?

Thanks again.

We used XL1-Blue competent cells for transforming large constructs. Unless otherwise specified, I think one shot TOP10 chemically competent cells should be fine for transforming large constructs.

As posted by many responders in many ways, just about any competent E. coli should serve your purpose more than amply, unless you happen to be putting together a combination that is inherently incompatible. 

I am assuming that you are cloning your DNA by ligation - that is where you need to begin to troubleshooting.

Are you using cohesive ends for ligation or blunt ends?

Is it a single insert or are you putting together multiple pieces of DNA in one reaction (we often manage as many 4 pieces in a single reaction in correct place and orientation)?

How have you ascertained that your ligation reaction is working?

Are you not getting any colonies on your selective media plate or relatively few?

Are you getting many colonies but none of them have your DNA insert?

Hi,

Thank you Venkateswarlu Kanamarlapuid. I will keep XL1-Blue competent cells as a suggestion. 

Tausif Alam, first of all thank you for taking the time to help me. I think you are absolutely right and we have to also tried to look at the ligation. I do think that the ligation is what is not working. However I wanted to make sure that it was not simple things like using the wrong bacteria or the vector being incapable of accepting such a large insert (why I asked these questions). 

I really appreciate further suggestion on how to improve the ligation. This is what I know so far;

• We are using cohesive ends, but only one restriction site (BamHI). However Im minimizing religation with alkaline phosphatase treatment of my vectors.
• It is a single insert, however a big one of 5.6kb.
• I have not ascertained that the ligation reaction is working before transformation, can this be done? (I think that it is not working).
• I have tried three different ligation kits: TOPO TA-cloning (life technologies), Fast DNA ligation kit (Epicentre) and lastly TaKaRa's DNA ligation kit LONG. 
• I get non or a few colonies, non of the colonies has my insert, it is only religated vector.

Do you have any suggestions for an alternative ligation strategy? It seems like my gene is a hard one to clone?

Hi Sanna,

I would suggest the following strategies to improve your chance of cloning a large insert into pEGFPC1

1. Increase the insert amount, if possible, in the ligation reaction.

2. Try to use directional cloning, which not only bypass the requirement of alkaline phosphatase treatment (can effect ligation) but also increase your chance of getting the insert in right orientation. Re-PCR your insert with BamHI site at the 5' end and no restriction endonuclease site at the 3' end. Digest the insert with BamHI and clone into pEGFPC1 digested with Bgl2 (compatible with BamHI digested end in ligation) and SmaI (blunt end).

3. If you still find difficulty in cloning the insert into pEGFC1, please use the ligation free cloning kit available from NEB or some other company.

Sanna,

You can check the degree of how well ligation worked by running the ligated products on a gel.

Dephosphorylation of vector portion, though a good idea in principle, often leads to issues that you seem to be having. This is primarily because the phosphatase enzyme is often not fully inactivated. Commonly used Calf intestine phosphatase is quite heat stable and requires Phenol-Chloforrm extraction, followed by precipitation of DNA for eliminating residual phenol, which if present, will interfere with ligation.

Other possibility is to denature the enzyme fully by using high concentration of chaotropic agents, such as guanidinium hydrochloride or guanidinium isothiocyanate. PCR purification kits sold by Qiagen and perhaps others, are based on this priciple. The DNA is retained by the matrix in the presence of chaotropic agent, the matrix is washed thoroughly to remove the "binding buffer" an deluted by water or Tris buffer. Avoid too much EDTA at thisi stahe, which may interfere with your next step, the ligation.

There are various other versions of phosphatases in the market that are claimed to be heat labile and do not require tedious cleanup, such Arctic phosphatase from NEB and Shrimp phsophatase, also from NEB.. You may wish to give that try. However, I cannot unreservedly recommend the former (Arctic), which in our hands was garbage years ago.

The reality is that what you are trying to do is a very simple task and you can skip phosphatase treatment of your vector. All you need is clean vector and insert (gel purified), add the insert in 3-5x molar excess, do a quick ligation at room temperature (~15min) in the presence of PEG and you are done!

In 40-50µl competent cells do not add more than 1µl ligation mixture and you ought to get hundreds of colonies on Amp plate. If you were to pick 20 colonies, some will have insert in one or the other orientation.

Best,

I would still dephosphorylate the backbone with Shrimp Alkaline Phosphatase (NEB) as good practice when performing ligations. You may wish to perform a number of different insert:backbone molar concentration ratios, as optimal concentrations can vary. I usually run 3:1, 6:1 and 10:1. And try running your ligation overnight at 16deg with T4 ligase (NEB) rather than the kits.

Venkateswarlu Kanamarlapudi, Tausif Alam and Dario Gerace, thank you so much for taking the time to answer my question(s).

I will do ligation without dephosphorylation, check my ligationproduct on gel, also use Shrimp alkaline in the future.

Tausif, "a simple task" is what I also called this project 14 months ago - but now om not so sure anymore. My other idea is to do the gene in small parts with Gibson assebly and see if I can get that to work. Thank you again! I will let you know how it goes!

Best,

Sanna Gudmundsson

Hi Sanna,

When we occasionally have problem with ligation, we use quick-fusion cloning kit from Biomake (http://www.bimake.com/product/quick-fusion-cloning-kit.html) for cloning. So, you may want to try the same kit when you want to try a different procedure for your cloning.

Sana, is it possible that the nature of DNA you are attempting to clone is the culprit?

We both know that at the face of it, what you are trying to do should take no more than a week, 2 weeks at the most.

Thank you Venkateswarlu Kanamarlapudi!

Dear Tausif Alam, I do think you are right! Im considering Gibson assembly to insert one part (800bp) at a time to see if I could escape the problem that way - what do you think about that?

Best, Sanna