Hi, I´ve had the same problems as you. The most Important considerations that you must keep in mind are the amount and quality of DNA before ligation reaction. Almost all ligases uses a ratio 3:1 of pmol ends to be successful and It works very well (See image). I rarely dephosphorylate the vector and I've gotten correct colonies (only when you want cloning fragments, It is not suitable for expression, because with a single digestion you can obtain bidirectional products). If you can get your fragment by PCR and after digest it, the result is better and easier. Other trick I use is for purification, I take a sample of my vector digestion reaction (>50 ng) and I do a gel electrophoresis to see quality and completly digestion, after that, I purify the vector digestion reaction with PCR or Plasmid purification kit, because the gel purification kit normaly has low yield recovery. I hope this information will be useable. Dont forget use controls if it is posible.

I have a few suggestions that may or may not help you.

1. A 9.8 kb plasmid is getting quite large.  If you're doing heat shock transformations, it can be difficult to transform plasmids over 10kb...and yours is quite close.  If you have access to an electroporator, I'd try that.

2.  I'd recommend using antarctic phosphatase instead of CIP.  That way you can save yourself from having to use the gel extraction kit and also exposing your DNA to UV light (which can cause all kinds of problems for gene cloning) by just heat inactivating both SalI and Antarctic Phosphatase.

in my history of cloning, failures never were due to a wrong ratio of vector to insert. it was almost always caused by fragments that were not perfect in one way or another. the most likely cause of your problem is the preparation of the vector. there is always a number of vector molecules that are not cut, and they will comigrate with your linearized vector on the gel.  the only way to make things better is to digest only a small amount of vector DNA (try 100, 200 and 500 ng) and use an agarose gel that has a long separation  way. digest and dephosphorylate as before and then run the three vector digests in adjacent lanes on the gel. run the gel (0.8 percent agarose or less) for as long as possible (heating development may be a problem). then sharply excise the vector band that has the nicest appearance in the gel. isolate DNA and use this for ligation. if the problem persists and additional sites for cloning are available I would rather prefer a strategy using two different enzymes. 

Please let us know how you solved it.

Best wishes, J Stolz

Hi Elena,

In addition to the great suggestions mentioned above, I want to say that some cloned sequences may not be tolerated by the host E. coli strain, increasing the background of cells with the empty vector. Check the target sequence for strong E. coli promoters and for inverted repeats. If the protein expressed by the cloned sequence is toxic to E. coli, try using promoters that have low level expression or that are inducible with tight regulation. Also consider using a low copy number plasmid as a cloning vehicle.

Try growing the cells at a lower temperature (30°C or room temperature). Colonies will take longer to appear. Try a different strain that tolerates repeated sequences (e.g., Stbl2 E. coli).

Good luck

Hello Elena

Many useful references on attachment 1

https://www.protocol-online.org/biology-forums-2/cloning.html

2

https://www.protocol-online.org/biology-forums/cloning.html

3

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&uact=8&ved=0ahUKEwjunbeTy-bVAhVhQpoKHfEFBCgQFgg2MAM&url=https%3A%2F%2Fwww.neb.com%2Ftools-and-resources%2Ftroubleshooting-guides%2Ftroubleshooting-guide-for-cloning&usg=AFQjCNGafycyVYsnFvWKlOq_dtoYthsmNw

4

https://www.rf-cloning.org/fluxbb/viewtopic.php?id=24

5

https://www.addgene.org/protocols/pcr-cloning/

6

https://www.addgene.org/protocols/lic/

7

https://tol2kit.genetics.utah.edu/index.php/Protocols

8

http://www.biotechniques.com/BiotechniquesJournal/2011/November/Cloning-efficiency/biotechniques-323274.html

9

http://www.biotechniques.com/BiotechniquesJournal/2014/April/Gateway-Cloning/biotechniques-351239.html

10

https://camelot.bioc.cam.ac.uk/~marko/methods/cloning.pdf

11

https://www.thermofisher.com/iq/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/cloning/cloning-troubleshooting-guide.html

12

https://web.mit.edu/jacks-lab/protocols/pSico.html

13

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361335/

14

https://2015.igem.org/wiki/images/9/9a/TU_Eindhoven_The_Cloning_Guide.pdf

15

https://www.nature.com/articles/srep00415

16

https://portals.broadinstitute.org/gpp/public/dir/download?dirpath=protocols/production&filename=cloning_of_oligos_for_sgRNA_shRNA_sept2015.pdf

17

http://dharmacon.gelifesciences.com/resources/faqs/what-protocol-cloning-own-hairpin-design-pgipz-empty-vector/

18

https://www.oxfordgenetics.com/SiteContent/TeamResources/core-cloning-protocols

19

https://functionalgenomicsfacility.org/media/FAQ_shRNA.pdf

20

https://www.plantphysiol.org/content/145/4/1161

Hi everyone,

Thank you for help. While struggling with cloning I came up with one important consideration. The fact that I am having colonies in empty vector ligation might be due to 1) poor dephosphorylation 2) incomplete digestion so I am bringing some of the uncut vector with me that will be very well transformed. I was suggested to run linearized vector overnight at 4 degrees so separate it properly from the uncut vector and it helps a lot! Still experiencing other problems but at least I solved this one!!

Thanks again I appreciate your suggestions!