Hi i'm hoping some of you might have some suggestions. i am trying to clone a small pcr product <200bp into the EcoR1/Sal1 site of the vector pGBKT7. The pcr product has 4 bp overhang after the restriction enzyme recognition site, so i should nt expect a cutting problem? For ligations i have tried a range of Vector to insert ratio s, to no avail. I have tried dephosporylating the vector. I have heard that Sal1 can be a difficult enzyme to work with? Are small inserts more difficult to clone?

did you get colonies?
which buffer did you use? double digest?

The following web site is a useful resource for determining how much overhang should be left for your enzmes to cut efficiently

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp

I would say you should cut with Sal 1 first, clean up the fragment and linearized vector and then cut in a 2nd separte step with Eco RI. And dephos the vector after each cut.

A 3x molar ratio of insert to vector always works well for me. pGBKT7 is 7.3 kb and your fragment is 0.2kb.

That means if for example your purified opened vector and your 200 bp fragment were both at 1ug/ul, 1ul of your 200bp insert will have 36.5x more molecules than 1ul of opened vector.

You should therefore dilute the 200bp insert stock 12.17x and combine an equal volume of insert and vector to get a 3:1 molar ratio.

Also make sure you have aliquoted the buffer for your T4 DNA ligase the first time you thaw it after purchasing it. It contains ATP and will rapidly go off with repeated freeze thaw cycles.

If you still can't get it to work, Private Message me. I have a protocol for insertion of fragemnts into any site in a vector by PCR that does not use restriction enzymes. I have dropped in fragments as small as 100bp and as large as 1.7kb.

Ya Thanks for the tips i have tried the sequential digest in addition to the double digest, for the double digest i usually use promrga buffer D but have tried REACT also. We routinely aliquot the ligase buffer so i dont think it is that. It has worked in the past for these vectors cut with these enzymes so it is more frustrating now that it wont work particularly when i hav nt changed anything that dramatically. I am presently trying to blunt end clone in the troublesome inserts and then subclone them into the vector if that does nt work i might be back to you guys. Thanks again for the tips

Triple check your sequences and scrutinize your vecotr for additional restriction sites - especially if you inherited it from a less than trustworty site. Just because a restriction site is listed in the poly-linker doesn't mean it is unique (oh the many lab hours lost to trust!)

Sal I has a bad rap, mostly becasue of methylation issue with genomic DNA. However, supercoiled plasmids do require a ten fold over digestion with SalI.

A 4bp overhang should be OK, but longer is always better!

I realise this post comes a year too late but I think it might be helpful for others who have similar problems:

Cutting close to the end of PCR products is never as easy as you expect. I would always recommend inserting PCR products into TOPO cloning vectors (TA or blunt, depending on whether the polymerase was proof reading or not). From there you can pick plasmid clones and get the sequenced easily and cheaply. When the sequence checks out OK then you can use the RE sites incorporated into the ends of the PCR primers, but now they will cut much more efficiently because they are not at the ends AND the plasmid prep is much cleaner than the PCR mixture. When you sequence a PCR product you can not be sure that all molecules are the same so despite getting a positive sequence back, the molecule that actually gets cloned into your plasmid of interest might be different.

If you want to cut PCR products and ligate them directly then I would always do a control ligation in which there is no vector DNA and about 5 times more cut PCR insert DNA than you would normally use in a ligation. Following ligation, run the whole reaction on a gel and see how efficiently the inserts multimerise. If you only get one band then neither end was digested well. Two bands indicates that one end digested well or that one end didn't ligate efficiently perhaps through being blunt. A ladder of bands is what you want.

Yes, you are right. SalI is not quite straightforward like most enzymes. If you check NEB catalog, you will find Sal I doesn't cut supercoiled plasmid well. Therefore, linearize your plasmid first with the first enzyme then cut with SalI.

By the way, I recently tried a new invention BioMagic gel-free cloning kit from a company. They claim you can clone any insert/vector of your choice without agarose gel with their kits. It sounds too good to be true. But I tried it anyway. I am amazed it works. Imagine cloning without running gel or gel purification. It is pretty cool.

For those of you interested, here is some more information and a link to the product mariners1984 referred to:

This product is manufactured by BioEfficiency Solutions, Inc. and here is the statement from their website:

BioMagicTM Cloning Products allow you to clone smart not hard!

Our innovative BioMagicTM Gel-Free Cloning Kits rely on our proprietary Gel-Free Technology. We developed a mix of proprietary chemicals, polymers, proteins and etc to specifically "tag" fragments of interest for cloning. The "tag" is based on certain cloning sites, length, adjacent bases, GC content, secondary structure and etc. After being "tagged", only fragments of interest are suitable for cloning reaction. In the meanwhile, undesired fragments become supercoiled at various level. The necessity of gel to separate the desired from the undesired is eliminated. Additionally, our products solve another common problem: cloning fragments inseparable from multiple similar-size fragments on gel generated due to no or minimal size difference.


Here is the product page link - BioMagicTM Cloning Products