Hi there,

If you get clones from control DNA then there is something wrong with your plasmid prep (have you checked the content of your DNA sample on gel?). Try the same transformation protocol with an other miniprep.

If transformation is made using a ligation mix, the problem might be more complex (digest efficiency of plasmid and insert, ratio insert/vector used for ligation...)

Hii.. Dominique Liger ..

Yes..after digestion i analysed the plasmid, pET28a on gel..I found that almost same size band (approx. 5.1kb) as two RE's are 72bp away from each other, and same RE sites were cleaved from the insert DNA .

For ligation, i used separate ligation components. As per kit, i used approx. 50ng prepared pET28a and 0.2pmol insert DNA (approx 75ng). Ligation was carried for 14hrs at 16 degrees.  

Hi again,

The ratio you are using is roughly 7:1 which should be OK. As you don't get any clone, the vector stays open during ligation. 2 possibilities : your ligase is not working properly and/or the circularization can't occur because of uncompatibility of DNA ends. What I would do is to test RE individually on the plasmid to check digestion efficiency (using the conditions you have already used for double digest) and as the two sites are close I would go for sequential digestion of the vector (you can't be sure about efficiency of the double digest unless you are able to spot the 72bp fragment on gel).

If you have to come to check ligase efficiency, do it on plasmid linearized by single digest and compare the number of clones obtained with the same amount of plasmid treated with or without ligase.

Thanks ..Dominique Liger

What type of competent cells are you using? How much DNA are you adding to the transformation? Unless you use the BL21 (DE3) Gold strain, the transformation efficiency of pET expression strains tends to be quite low. You might be more successful with an initial transformation into a cloning strain (e.g. DH5a, JM109) and then screening for recombinant plasmid before moving to your expression strain.

Hello..Daniel M Cohen ..

I used Novablue for initial cloning host fro transformation. For the ligation experiment, i used 50ng prepared pET28a and 75ng (0.2pmole) prepared insert. After ligation, i used 1ul undiluted and 1ul (1:5 diluted) ligation product for 20ul of competant cells and transformed as per the protocol using heat shock method at 42 degree and Upon half an hr incubation at 37 degree, plated onto LB agar plates containing 30ug/ml kanamycin.

The kanamycin resistance enzyme can take up to an hour to be expressed. You should get better results by waiting longer to plate your cells. Using NovaBlue, I'd expect good results if you plate 1/4 to 1/3rd of your transformation reaction. Make sure you have fresh ATP in your ligation buffer.

If this still doesn't work, then something is wrong with the ligation. What is the size of your insert? How are you performing your restriction digest and with which enzymes? Are you gel isolating your DNA? Too much UV exposure or too lengthy a restriction digest can damage your DNA rendering it either unable to ligate or unable to propagate in E. coli. These issues are the most common culprits in failed ligations.

Thanks Daniel M Cohen for the suggestions..

The insert size is 1.2kb...This insert was digested using NdeI and HindII-HF using Cutsmart buffer. After 3hr (as mentioned in kit protocol, 2-4 hr) digestion at 37 degrees, the digested product was purified using GenElute Gel extraction kit , Sigma and care was taken while cutting the DNA band for long exposure of UV..

Hi Yogesh,

i did not get how you amplified your cellulase sequence. Do you have it already in other plamid? Or are you trying to amplify it from the original organism? When you do the amplification of cellulase you have a clear and unique band on gel? If yes you do not need to run it on gel. Do the purification directlly from the PCR product (the yield is much higher).

Regarding digestion, once again you don't need to run it on gel, even your pET28 digestion, because both RE are close and the fragment that will be remove is short and will be removed during purification.

For clonning, you need to use a 3:1 ratio (insert:vector). I do a final volume of 20uL and use bacteria (DH5a or simillar) high competent (>10^6), do not use expression strains.

I do the T4 reaction at 24ºC for 10 min. Use all (20uL) to transform bacteria and plate all.

Good luck

Hii..Andre Miguel Cordeiro..

The DNA insert is in another plasmid (pGEMT-easy) which is been amplified using the degenerate primers for the particular sequence..I have purified the insert using Gel extraction because PCR clean up will not work as some traces of unutilised plasmid template might be there, which may interfere in subsequent steps..

Thanks for the suggestions..

double check the insert (digested by two enzyme or just the PCR frgment),check the ligase,or the efficiency of the competent cell(TOP10>DH5X>BL21), check the ATP in ligase buffer all these will effect the cloning efficiency.

Hey Yogesh, 

I'm doing cloning experiments in pET28a with a 2kb fragment (AT-domains) and transformed them into E. coli BL21(DE3) (NEB) competent cells. Maybe try different competent cells.

Is your fragment maybe toxic for the cell? 

Thanks Min Li.

Hii..Henrike Miess ..

May be the the fragment is toxic to the initial cloning host, Novablue..If this is the case then what will the initial cloning host rather than Novablue...??

Another question, how long did you cultivate the plates sometimes it takes up to 3 days.

I red the protocol, and I was wondering why the incubation before plating is just 30 min, because from my experience it is essential to incubate it for 60 min for expression of the  resistance gene. 

@ Henrike Miess.

Same question arised to me..In 30 minutes incubation, i didn't find any colonies in first experiment then i repeated the experiment with some increased amount of ligase and 1 hr incubation..now i quite satisfied i got some colonies..

I think the 60 min are def. necessary for a good colony yield. Good luck with further experiments, if you are curious you can repeat it with the old ligase amount and 60 min incubation and the other way around. Would be an interesting thing.

Thanks Henrike Miess for the interaction..

@ Rubina Mushtaq..

Yess..Rubina..I had deactivated the ligase by incubating at high temperature..