for ligation you can use 3 different concentration of your vector and gene i.e 1:1, 1:2 and 1:3 (50ng of vector). transformation with pCambia in difficult.

just check few things

1) digetion and elution is proper.

2) antibody kanamycin should prepare freshly  and add once media reaches approx 400C becouse its heat sensitive also.

3) and your competent cells are working fine or prepare it freshly.

May i know how u are doing transformation by heatshock method or electroporation.

becouse pCambia ib around 10 kb length its quite difficult with hwatshock method 

Hello Zaw Ko Latt

First I would like to ask you how big is your fragment FatB?, I have succesfully ligated a 1.4kb fragment into pCAMBIA1302 and cloned into DH5a using the heatshock treatment so I will share my experience and I hope it will help you,

One of the main problems I had were my competent cells, just as Phani suggested above you should take into consideration all his tips, specially competent cells and antibiotic treatment.

I was trying to use competent Top10 E. coli but my cells were not giving me any result, so I changed to Competent DH5a and it worked right away, make sure they are competent by using a transformation control with the pCAMBIA1302 mock (you should get a lot of colonies).

The other thing I tried was to use a small ligation reaction of 5 uL to maximize the concentration of both plasmid and insert, try to increase the concentration of your fragment, even if your plasmid gets a little low on concentration, in my case I used around 30 ng/uL of insert and 10 ng/uL of plasmid, remember that for big plasmids like pCAMBIA1302 it is recommended to use a 1:5 or 1:10 ratio.

Then do a small ligation reaction at 16°C O/N and use it to transform the cells. Here I would also suggest increasing the amount of cells (I use 50uL of competent cells at the highest OD600 possible for competence which I think is around 0.3 or 0.4).

Finally one trick that I use is the centrifugation of cells right before I plate them into the agar, do the whole heatshock treatment and growh and right before the plating step you centrifuge them at 1000 g for 5 to 10 minutes (very gentle centrifugation), then discard all but 100 uLof culture and use this 100uL to resuspend the cells and plate them into the agar, this increases the transformation efficiency.

Let me know if it helps.

Best of luck!

Thank you very much for your suggestion. The size of insert is 1.2 kb. After ligation, I transformed into competent E.coli DH5 alpha by heatshock method. Now I am trying to change the T4 DNA ligase. But my disappointment is that when I purified inset DNA after enzyme digestion, the concentration is very low (around 4-5 ng/ul). So I have to use much volume to get the desired concentration. 

In my case, for growth of E.coli after heatshock, it was allowed at 37C for 2 hr and centrifuged at 8000 rpm for 1 min. And pellet was resuspended in 100 ul culture and plated on agar. In positive control with uncut pCAMBIA 1302, they grew on LB containing 50mg/L kanamycin. One thing that I am afraid that the concentrations I use are not correct because the concentrations of FatB after purification from gel are very low. I really appreciated for your suggestions.

So 1.2 kb should be ok for ligating in this plasmid, I think your problem is on the dna concentration.

I was also having this problem and I did some changes which are suggested in some forums.

First of all, does your FatB fragment comes from a plasmid, or is it a PCR product, because I will advise not to purify it from the gel, if it is a PCR product then purifiy it using the kit directly, without any gels. just do the PCR to get the amplicon, then digest with the enzymes and finally purifiy it with the kit, it works so dont worry about using gels, are your enzymes from Thermoscientific or NEB? I see here that if the're from Thermo then you can use one buffer and the same T° for both, if they're from NEB then you can do the following, prepare the reaction as if it were for a double digestion, but dont add the BstEII yet leave it with the missing volume, first incubate for 2 hour at 37°C using BglII, after the 2 hours increase the T° to 60°C and add BstEII, then incubate for 2 more hours and purifiy directly with the kit, dont use the gels.

Which kit do you use to purify your gel fragments?, I'm using SV Wizard PCR clean up system from Promega. (https://www.promega.de/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/) and was getting around 8 to 9 ng/uL, then I used the following modifications and manage to obtain around 20 to 30 ng/uL from a gel purification, the first suggestion is to try and cut the gel as precisely as possible, trying not to get to much agarose as this disturbs the column, second I used more binding solution (1.5x times what's suggested) then make sure that the agarose is completely melted and then incubate it in the column for 10 minutes, then i Centrifuge as stated in the protocol and took the eluent and pass it trough the column for a second time and incubate for 1 minute, centrifugation again (16000g x 1 min), then the normal washing steps as in the protocols, and in the last part when you use water to obtain the DNA is the most important modification.

It worked really well for me, so when you start the purification procedure get a thermoblock or water bath at 70°C and put your nuclease free water to heat up till this Temperature, then when you are in the final step where you use water to free the DNA from the column, add 40 uL of the preheated nuclease free water into the column and incubate for around 10 minutes, then centrifuge normally at 16000g x 1 min and verify your concentration by nanodrop.

Then try the ligation settings I stated above using  a higher amount of the insert, try to use a 1:5 ratio (vector, insert), also verify that this 8000 rpms are not much for your cells as they're fragile from the heatshock process and this can significantly decrease their viability.

I'll attach an article I found about different competence and buffers, it helped me a lot during my experiments and is really nice.

Hope it helps

Alejandro Gomez Mejia, Thank you so much for your kind suggestion and taking considerations for my problems. Firstly, I tried to ligate FatB into pMD18-T and transformed into E.coli DH5alpha. To this step, it was OK. After extraction recombinant pMD18-T from transformants, recombinant pMD18-T and pCAMBIA1302 were digested with Bgl II and BstEII at 37C for one hour to create FatB and pCAMBIA1302 sticky ends, and checked bands and purified from the gel. These two enzymes are from Thermoscientific. At that time, the concentration of FatB was very low. So I have to use too much volume to get the desired concentration. I will try again to purify DNA from the gel to get high concentration. I think, your experience can solve my problem. I really appreciate your suggestion.·

Hi Alejandro Gomez Mejia, in the final step of DNA purification from gel, did you use warm water from 70C or didi you cool water before adding into tube?  

I used the water at 70°C but I got confused with the tube part or you mean the column?, because this mainly works for column based purification like the kit I mentioned above, there is some theoretical foundation that with an increased T° the DNA elutes more easily from the column and in my case I got to double the DNA concentraton from the purification, how did it go for you?

It appears that 16C may not be the optimum ligation temperature. Normally one would also see the buffers, but apparently there seems to be nothing wrong with those. May be 20C or 12C might do a good job. 

For my case, I taked out the water two or three minutes earlier from 70C before I used. So I would like to know that did you preheated water  immediately after taking out from 70C to add into column? And for gel melting, I put the tubes containing the gel in 50-55C for 10 minutes. And for you, how many minutes did you put? Here, all instructions from Kit are in Chinese Language. So I am afraid that I am wrong. Thank you.

Hello Jai Ghosh, Thank you so much for your kind suggestion. As your suggestion, I also need to study at different tempearture for ligation. This is one of the things to consider. Thank you.

Hello, what I did was that I got the preheated water just before I added into the column for elution so it was at 70°C then incubate for 10 minutes and centrifuge, and for the gel melting 55°C for 10 minutes is the recommended time and T° but you should always check that the gel has completely melted so there shouldn be blocks or pieces of the gel in the tube. Tell me if it works, also one more thing you can try is doing a big digestion (50 to 60uL) and add it all to the gel in one well then purify it.

Alejandro Gomez Mejia, Thank you so much for your kind suggestion and answers.

Hi, finally i managed to get positive colonies for ligation. I changed T4 DNA ligase with new one from BioLabs, New England. The efficiency of previous one is becoming poor. The concentrations of vector and DNA insert I used were 23.45 ng and 37 ng.