Hi,
I am having troubles cloning a Cas9 gene (around 4300 kb) into a vector, replacing a LacZ cassette. (I use the uloop system.) I use SapI as a restriction enzyme and T4 as a ligase. I cloned several other constructs into the same kind of vector with high efficiency (mainly white colonies, and almost all carry the correct insert when screened).
When I try to insert Cas9 (or also dCas9) I yield the same number of colonies, with again almost all white. However, when I screen the colonies via colony PCR or restriction digest the isolated plasmids of the colonies, no vector carries the insert. I did not sequence these plasmids, but from the restriction digest it suggest, that the plasmid only lost its LacZ cassette and was closed again without any insert (eventhough overhangs are not compatible).
I tried two settings for GGC:
37 C 5min...............37 C 5min
16 C 5min................16 C 5min
(25cycles)................(40cycles)
65 20min-................65 20min
85 10min..................85 10min
4 hold......................4 hold
I tried two different buffer conditions:
-T4 buffer only
-Or 50% T4 buffer, 50% Cutsmart buffer
I started with fresh Cas9 (and dCas9) PCR templates and with several fresh receiver plasmids. Also I used different competent cell batches.
I double checked the overhangs of receiver plasmids and the overhangs created on the Cas9 insert.
I am running out of ideas..
Does anyone know if GGC with single large inserts (4000 kb+) effect GGC efficiency?
Does anyone have a suggestion how I could improve GGC efficiency?
Further troubleshooting suggestions?
I really would appreciate your ideas!
Thanks,
Florian
In my experience Golden Gate reaction has considerably (about 10-fold) lower efficiency with PCR products vs plasmids. First and foremost, it must be assured that PCR product is pure (spectrophotometry check). Second, does concentration for the insert is calculated using moles (not mass). If same mass is added to the reaction, large inserts can end up at very low molar concentration. Third, are overhangs indeed ok. It is always worth to re-check this by using tools for in silico cloning. For example, Benchling allows one to choose fragment and then perform in silico GG reaction to check that overhangs are ok. I know that it was mentioned that overhangs were checked, but due to high-throughput nature of GG reaction, frequently, errors here are over-looked. Fourth, also from this category. Is the antibiotic on plates correct?
In my program for GG cloning cycles are (1min - 37C, 3min - 16C) X25. Using longer cycles seems unnecessary, as it appears that even under those conditions the vast majority of plasmids are ligated by 10th cycle. Prolonged incubation at 37C might decrease enzyme activity and be detrimental. But I don't think this is the problem.
If none of these seems like an answer I would adivise to titrate the amount of Cas9 amplicon; i.e., set up several reactions with increasing amount of the insert.
There is also an option of 'classical cloning'. Just precut plasmid and insert with restrictase, purify both and set up ordinary ligation reaction.
If cloning absolutely fails, the most likely reason is wrong overhangs. In this case overhangs shall be checked by sequencing. The insert can be TA-cloned for this.
And TA-cloning is still another option. It is possible to TA-clone the insert, purify the plasmid and then use this plasmid to transfer the insert from the TA-plasmid to GG plasmid.
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