Hi there,

How far are the 2 sites in the recipient plasmid? If too close, enzymes might interfere in a double digest reaction and reduce the probability to get the plasmid fully double digested. If it is the case, sequential digest should be preferred.

How much plasmid do you engage in the ligation mix? It shouldn't be more than 100ng... What molar ratio do you use between insert and vector? I should be 3 to 5...

No ligation product purification is required.

Blunt ends need more ligase for efficient ligation (see NEB leaflet and stick to the amount required for blunt ends)

You should systematically run 2 controls:

vector alone without ligase (to see if intact plasmid is present)

vector alone with ligase (to see if more clones are obtained due to selfligation only).

In your case, you only know there is not any intact plasmid as you don't get any clone.

I am facing the same problem. I have a codon-optimized gene cloned into pUC57 with AvrII and XmnI overhangs. I want to subclone the gene into a yeast expression vector (pKOV410). The size of my gene is 1429 bp and the expression vector is 6352 bp. The overhangs are close to each other on the vector. I did a doble digest on the pUC57 to release my gene, ran a gel and extract my gene of interest using Promega gel extraction kit with a final elution volume of 50 ul. I get 20ng/ul concentration of my gene. I also did a double digest of the pKOV410, run and extract the linearised vector. The nanodrop concetration of the vector is 45ng/ul. I am using NEB ligation kit. I tried ligation at different environment (3hr RT, 16hr O/N, 4 degrees O/N). I take at least 3-5 ul of my ligation into 50ul JM109 competent cells which we just bought. I do a control with uncut vector and i get hundreds to thousands of colonies. The ligation kit is new and i made aliquots of the buffer the day i received the kit. The enzymes are from NEB & they recommend digestion for 5-15min, but it seams not to be enough, so i went for 1-3 hours, that works well. But i still dont get a single colony. One of the overhangs is blunt & the other is sticky. I have not tried sequential digest yet. The antibiotic resistance for the expression vector is Kanamycin. I have tried different ligation ratios (1-20) but NO colonies.

ANYONE WITH SUGGESTIONS THAT CAN HELP ME GET PASS THIS STEP?

Well you do not need to perform clean up after ligation. You could increase the molar ratio of the vector to insert to 1:6. Then, you could try for 4 degree ligation, i have found it several times that the ligation could work better even though the standard is 16 degree celsius. Although I am yet to find any valid reason for this apart from the obvious decrease in the movement of DNA that could help in ligation. Apart from this, you could also add extra ATPs in the mixture if possible so as to increase the activity of the ligase. As for control, you could make another reactions containing single digested vector of both EcoR I and MFeI so as to see the optimum time requirement for the ligation.

Here's the ligation calculator:

http://www.insilico.uni-duesseldorf.de/Lig_Input.html

Hope this helpful to both of the questioners

Iason Karyofyllis If you are using NEB enzymes that comes with the Cutsmart buffer, i would suggest you digest both your plasmid & gene overnight, run a gel and purify. Do ligation and leave that on your bench overnight. Apparently Blunt & sticky ended genes works best that way. Then use about 1-5 ul of your ligation for transformation in 50 ul competent cells. I was having the same problem, but after following the protocol i just mentioned, i got colonies for the first time after months of struggle. Good luck!