Hi there,

I always prefer to run digestion on purified PCR product in parallel with the one of the vector so that the latter serves as an activity control to the former. If sites are close in the vector both RE may compete for binding resulting in lower digest yield. To avoid this, sequential digest is recommended.

T4 ligase is the usual ligase for cloning purpose.

What are the bands you are talking about? Are they about ligation mix or else?

I agree with Dominique about the sequential digestion. In addition, have you double checked if either of your enzymes are sensitive to Dam/Dcm methylation?

Hi,

Are you dephosphorylating the Vector? I always do with Shrimp Alkaline Phosphatase (rSAP) . This will inhibit self ligation. When doing your transformation always include a vector only control.

After PCR , clean the amplicon before cuting with your restriction enzymes.

Then run a gel to see the producl. Gel purify and ligate.

After digeting the vector also run a gel and purify your vector.

When using T4 DNA ligase, I always digest overnight starting at 4 degrees and slowly letting the reaction increase up to 16 degrees.

When doing transformation , only use 1/10 of the ligation volume. I usually do 20 ul of stbl3 bacteria and 2 ul of ligation.

Thanks and let me know

Angel

Although not a solution to your question directly, another potential solution is using a different vector system. If you're amplifying with Taq, the TOPO-TA cloning kit is very simple and efficient - usually a go to of mine.

Dear Janine,

These an alternative to the kind a valuable comments offered above.

I started in the las 2 yeasr to shift from the regular ligation to ligase independent cloning (LIC) based in the Gibbs assembly method. Since then I haven't had any more problems of this type. Nowadays there are several available kits (I'm currently using the one from NEB) which aren't too expensive and I strongly recommend them:

https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf

https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Product%20Information

You will have to redesign your primers to add the appropriate overhangs matching your vector and the in a 15 min reactions. In general it takes 1.5 days from getting your plasmids to get your positive clon.

Hope it may help.

Hi,

I also forgot to tell you that when I have difficult ligations I heat inactivate the ligase at 65 degrees C for 10 minutes , follow by ice incubation and then transformation.

Thanks

I'm sorry I wrote in a little rush and I made some spelling mistakes. In the last part of my answer above I meant you can have your positive clone 1.5 days after your receive your primers. You have to redesign your primers, run the PCR, purify the PCR product and your digested plasmid and the reaction to ligate them can be as short as 10 minute with an efficiency above the 90%.

As Angel A. Rivera mentioned above, dephosphorylating the vectors will increase considerably the efficiency of cloning. For this I'm using regularly antarctic phosphatase.

https://www.neb.com/products/m0289-antarctic-phosphatase#Product%20Information

Good luck!

DPN1 disgest your pcr product (to get rid of the template)

purify your pcr product with mini column

if you have been carefull, you have added -nt upstream you pcr primer (i usually add 6 whatever the RE I am using, it helps)

digest for several hours (and do not heistate to digest overnight in an incubator or a pcr machine)

gel purify you PCR product and your digested destination vector

run an aliquot to calibrate the concentration

perform you ligation as usual

and basically...you will get your recombinant

Dear Janine wohlers

I agree with Angel. It is the protocol defined by Sambrook and Russell. Notable you can choose to have control alkaline phosphatase. It means you can culture alkaline phosphatase positive/negative ligated separately. However, my experience tells me ligation failure refers to competent cells. If you competent your cells yourself it is very important to choose wright calcium chloride. Also, all the process should be done on the ice instead the heat shock processing that must be done in 42C. Do not pipet your cells vigorously. Try different concentrations of your ligates but 2 or 3 ul is better. Try different concentrations of your cells+ligates to culture according the size of your plate. According to your antibiotic selection marker you might have satellite colony. Do not use them.

Best Regards.

Dear Janine,

Besides all the good comments you have received I want to make a question and sugggestion.

I guess you are using two different REs, so you have to make a transformation control to verify that the plasmid is correctly restricted, that is you must transform bacteria with the ligation reaction witout insert (I mean plasmid, enzime, buffer but no insert). That might be one big problem

Hi

I prefer purified pcr product before digestion. I use 1 Units of restriction enzyme for 1ug of DNA for 2 hours 37 °C, inactivate the enzyme and then purified the products. for reaction of ligation i use T4 NEB, 400 U for reaction all nigth 16°C, inactivate the liagasa 65°C 1 0 minutes , follow by ice incubation, you can run 4 ul of ligation for checkin the reaction, Check this web site for calculate your liagasa reaction and you can use 100ng of vector

good luck!!

https://nebiocalculator.neb.com/#!/ligation