I have noticed that certain restriction enzymes are far less effective (or inactive) in T4 DNA ligase buffer. I would try using the buffer supplied with your enzyme with the addition of ATP to 0.1 mM (required for function of T4 DNA ligase). T4 ligase tends to be way less picky about buffer conditions than restriction enzymes.

I have not personally done Golden Gate cloning before, but from what I understand your thermocycling conditions may be able to be reduced since you do not appear to be preparing a library with this method. An hour at 37oC followed by heat inactivation (5 minutes at 50oC and 80oC each) may be sufficient. Cycling repeatedly between 37oC and 16oC for 5 minutes each, followed by the heat inactivation step may also work. I would first start with the buffer, but you may be able to reduce the amount of time needed for incubation which may help speed along any further optimization.

Tyler and Niklas - thank you for your feedback. I agree that the buffer is the most likely problem leading to inefficient cutting. I'll post back with what works/doesnt work for me just as a future reference.

Jason,

I have same problem. Hope to hear how you optimized your cloning.

Just to follow-up on this question from a long time ago with what I have found to work.

1. I think the major underlying issue is that many of the vectors (eg px330) have a very small sequence of DNA that is excised and the restriction enzyme cleavage sites are very close. I believe that there is significant hindrance as a result which significantly limits golden gate cloning efficiency. What I have done for these vectors which has been very successful is to clone in a LacZ reporter sequence into this site (using standard gel extraction/ligation cloning). This gives me a vector that I can always use in the future for golden gate cloning, and allows for blue/white screening of colonies. Using the protocol below I have achieved 100% success rate when picking white colonies (>90% of the plate are white) although I haven't systematically looked at this. I think this approach of adding a LacZ reporter site between the golden gate recognition sites makes your experiments much easier down the road, especially if you are going to clone into this vector several times.

2. I have switched to the Thermo enzymes, as I do not believe that the enzyme offerings for golden gate cloning are optimal from NEB (although I love all of their other enzymes). Purchase the fast digest versions.

3. Do not use the digest buffer, but instead use the T4 ligase buffer. This solves several issues as it maximizes the efficiency of the T4 ligase and the restriction enzymes perform well in this buffer. Also, Esp3I which is frequently used requires DTT which is present in the T4 ligase buffer but is not in the fast digest buffer - so no need to supplement.

4. T4 ligase - there is a low concentration and a high concentration mixture available from NEB. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning. I haven't tested this myself but use the high-concentration for multiple insert cloning.

5. Cycling conditions - the article mentioned in #4 also found that there is increased efficiency when cycling between 37C and 16C but I'm not so sure that this matters. For single insert cloning incubating at 37 for 30 minutes is plenty, and I use 6h or greater at 37 for multiple insert cloning. T4 is actually more efficient at 37, but the idea of cycling is that 16C promotes sticky-end annealing. I just use 37C but have done much less multiple insert cloning than single insert cloning.

I strongly recommend inserting a LacZ reporter into your cloning site for your vector as this increases the distance between restriction enzyme sites and makes colony selection a breeze. I have not systematically tested the other suggestions, but these are my general feelings on other factors that might help.

General protocol for cloning into a vector such as px330

Anneal oligos:

2ul 100uM F primer

2ul 100uM R primer

16ul H2O

2ul T4 ligase buffer

Ramp from 95 --> 25 at ~1C/min

Dilute oligos 1:200

1ul annealed oligos

199ul H2O

Cloning reaction

1ul dilute oligos

Xul vector (50-100ng)

H2O to 11.5ul

1.5 ul T4 ligase buffer

1 ul T4 ligase

1ul restriction enzyme

Total 15ul

Incubate at 37C for 30min

Transform 2ul of reaction on X-Gal/IPTG plates

Pick white colonies

recently I used golden gate as well. I get plenty of false positive (lots of white clones with no insertions)

your question I suppose is because of the amount of plasmids too high??