Hi all,
I'm currently in the process of constructing a number of plasmids from a series of interchangeable PCR-amplified constructs and, having done some reading, I am keen to try and apply the Golden Gate cloning method to accelerate the cloning process.
Having done some preliminary reading, I've designed my primer system and created my destination vector; however, I have a few technical/troubleshooting questions that I would greatly appreciate input on from people who have used this technique in the past.
1) I have read on one protocol that NEB-sourced BbsI (a.k.a. BpiI) exhibits extensive star activity; while I have not seen this in other digests I've done with it, has anyone experienced this problem during Golden Gate methods?
2) Has anyone experienced difficulties with end-filling (or 3' overhang nucleolysis) of sticky ends when performing Golden Gate cloning on PCR products? I plan to purify the products before digestion, but have read one or two questions on RG regarding this issue. Any guidance would be greatly appreciated.
3) How do you choose what number (and duration) of 37/16 degree cycles to use? I'm initially planning to try 10 cycles of (37-5' then 16-10') but this is a relatively arbitrary programme culled from other protocols; any suggestions regarding more guided programme design would be greatly welcomed.
4) Any other tips/tricks that I may not have mentioned or would benefit from are of course also welcome!
Thank you very much in advance for your help,
Regards,
Dan
I can only answer question 2) since I haven't used golden gate: there is no problem end-filling sticky ends. This is routinely done for any blunt end ligation. I use the Quick Blunting Kit (NEB) and now I plan to use just T4 DNA Polymerase to reduce costs. T4 is active in all NEB buffers, you just have to supplement with 100 µg/ml BSA and dNTPs.
Hi Soledad,
Thanks for your reply.
I'm afraid I wasn't completely clear in my question. The Golden Gate method requires an array of non-crosscomplementary sticky ends to coordinate concatenation of the cleaved constructs in the right order. As a result, I'm trying to PREVENT end-filling and overhang trimming, not encourage them; if they happen, the various ligations will no longer be directional and hence I'll get fragment shuffling.
My concern is that, through residual polymerase/exonuclease activities (or passive hydrolysis etc) I may lose the 5' sticky ends generated by BbsI, as per the question: https://www.researchgate.net/post/Problem_with_restriction_ligation_cloning-Fragments_ligate_without_the_proper_overhangs.
Cheers,
Dan
No problem. My only suggestion is that you start with a good amount of PCR product (I imagine you will gel purify and therefore lose a lot) and then use the minimum amount of enzyme and time for the digestion (any partial digested product will not be ligated anyway) and then go directly to the next steps. Sticky end ligation is very efficient and shouldn't present a problem. Did you already contact NEB tech service to discuss your questions? they are very helpful and knowledgeable.
Hi Soledad,
Thanks for your reply. The reason I am interested in applying the Golden Gate method is precisely because it is unnecessary to gel purify your cleaved PCR products, and there are no "next steps" to proceed to - it's a one-pot, one-step reaction in which all cleavages and (correct) ligations occur simultaneously and irreversibly. If you're interested in a more thorough description than I can provide here, check out PMID: 24395361.
I'm reasonably experienced with cloning, and thus am pretty familiar with most of the protocols for and pitfalls of most standard protocols. What I'm really looking for here is particular information on how standard cloning approaches/concepts may be altered within the GG protocol, and guidance regarding particular problems/complications other researchers have reported in the grey literature but which are absent from published material (for example, the reports by several labs of BbsI star activity, which NEB explicitly do not mention).
As it stands, I've assembled my blue/white-competent GG-compatible destination verctor, designed my overhang array and prepped my inserts; I'm just waiting for any final guidance re: GG specifics before I run my first iteration.
Cheers,
Dan
Hi Daniel, here are my suggestions/comments on your questions:
1) I haven't seen much trouble with BpiI golden gate cloning beyond normal cloning 'issues'. Usually, screening more than 4 clones from the reaction is enough to find the right one.
2) It seems that it's better to proceed to cloning after PCR and purification as PCR product may degrade over time. As for the actual cloning, I've seen that efficiency drops down big time when cloning PCR products (especially if stitching together more than 2) are used.
3) I usually use this program: 20 cycles of 37°C for 3:00, 16°C for 4:00, then 50° for 5:00, then 80°C for 5:00 and then 12° for ever...
Cheers,
Daniel
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