http://miller-lab.net/MillerLab/protocols/molecular-biology-and-cloning/gibson-assembly/

https://www.addgene.org/plasmid_protocols/gibson_assembly/

Here are some web links

http://miller-lab.net/MillerLab/protocols/molecular-biology-and-cloning/gibson-assembly/

https://www.addgene.org/plasmid_protocols/gibson_assembly/

Here are some web links

I also want to mention that I designed primers that amplify the vector without extension. This comes handy when I want to use the same backbone for different reaction. Did someone find difficulties using this same strategy in Gibson Assembly?

To answer your first question: if you are going to do Gibson assembly, you really need to use very high efficiency competent cells - these are going to have to be commercial (and expensive), unless you have someone in your institute who is _amazing_ at making competent cells. This means that the cells are the most expensive part of the process (as you can easily make the Gibson master mix go further by cutting reaction volumes). So, unless you plan to do an _awful_lot_ of Gibson cloning, I would say that buying the kit is probably a better use of resource. You'll waste quite a few competent cells optimising the homemade master mix (I did before I gave up), and a lot of time as well. The Gibson kit also contains a positive control, which is very helpful.

To answer the second question, I don't think that amplifying the vectors without extension is a problem. It just means that your primers for your inserts will be very long (I find that a 40 bp overlap is much more efficient than 30 bp). If you plan to make it a standard method for doing a lot of cloning, then having one half that you don't have to optimise each time will probably be very worthwhile.

Good luck!

Yes highly competent cells should be use...10exp8. 9..

The rbcl2 competent cells are easy to prepare...and cheap 10exp7 are routinely obtained...never used these for GA..

ALTERNATIVELY, prepare and calibrate elector competent cells, if you have an electroporator (but after your GA, DESALT YOUR MIX WITH BUTANOL EXTRACTION,,

At least for one or two inserts (haven't tried beyond that) homemade seems to work fine with our homemade competent cells (made according to http://openwetware.org/wiki/TOP10_chemically_competent_cells, but using DH5-alpha).

I am using a home made Gibson mix.  My fragments are the promoter (1kb), gene (2kb) and ter (250bp) that need to be asseble in a backbone vector (2kb). Whenever I do the Gibson assembly and transformation, I always get colonies. But whenever I check the colony, it only contains the vector... Does anyone know why it is always the case happening to me?thanks!

Hi @Christian Cantos,

I'm using the protocol from Miller's lab! See above. And it works nicely. Make sure you do the DpnI treatment this is to digest any wild type plasmid in your reaction.

Briefly:

I PCR amplify every fragment (20 bp overlap hangs on inserts only, this way I can use the same primers for the vector every time I need it), 1 hr DpnI (NEB) treatment (0.5µL in 30 µL reaction) at 37 degrees, column clean up all fragment. While DpnI reaction is taking place I run 5µL on gel to see whether PCR products with expected sizes are amplified. I use 2X of each inserts! And this works for me! Also take along a control by mixing 15µ water with same amount of DNA material as in your GA mix. Transfer both in TB10 ( I think any other strain could be used) high competent homemade cells. You need high competent cells for this to work.

Cheers

TS.

Hi, can I change the Taq Ligase with T4 ligase in the recipie. I know there are certain differences in both. One uses ATP other the NAD.

No you cannot! See below! Or NEB Website

T4 DNA ligase atalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids (1). T4 DNA ligase will seal nicks for these DNA substrates.

Taq DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini of two adjacent oligonucleotides which are hybridized to a complementary target DNA. The ligation will occur only if the oligonucleotides are perfectly paired to the complementary target DNA and have no gaps between them; therefore, a single-base substitution can be detected.

If you are following isothermal protocol and keep your reaction at 50C  then T4 ligase will be heat inactivated quickly. Taq ligase is stable at 50C and will continue doing its job. 

I made the cocktail and tried to insert about 1 Kb fragment in the pUC 19 MCS site. I used 1:1 and 1:2 of the vector to insert. The amount of DNA used was 100 ng in the 20uL assembly. The overlap/homology was 17 nt long. I transformed them in DH10B electrocompetent bacteria. I have got multiple colonies all blue on the IPTG/Xgal meaning no insertion I still have to make a colony PCR but I think the colony PCR is pointless as all have the intact LacZ gene. What kind of troubleshooting should I follow. Any suggestions ? 

Try cutting the MCS with a restriction enzyme and gel-purify the cut vector.  This will reduce your background.  I estimate the concentration of vector and insert by gel electrophoresis alongside a DNA mass ladder.  UV spectroscopy is not reliable at the [DNA] usually obtained during the cloning process.  Does the control work?

Also, I looked into making the cocktails versus just buying a kit.   Unless you are planning on doing a lot of genetics, the cost is quite high to make the cocktail yourself.  If the cost is spread over your entire lab, it might be acceptable.  Also the kits come with a control to show the reaction is working.

Does any one have an idea about which one is the best for cloning of larger fragments (10-12 kb) into BAC vector, either using Gibson Assembly kit from NEB or In-fusion HD cloning plus from TakaRa Clontech?

For how long does Gibson assembly master mix works efficiently? I have a year old preperation of Gibson master mix and I am not able to clone my fragment. 

BTW, our lab makes Mix-and-Go competent cells using the zymo transformation buffers (no conflict). They're easy to make, will work with gibson cloning and are really cheap (50mL of ultracompetent cells for $100. We grow them up with zymobroth). Just add 200uL of competent cells to the Gibson mix and plate immediately. Photos compare 2uL Gibson mix into ultracompetent cells using NEB protocol vs 8 uL added to 200uL of homemade bugs and plated immediately. 

Hi every body

I am using this kit but I am trying just to assemble the fragments then clone the resultant fragment in plasmid using TA cloning. Is this applicable?