pCAMBIA1302 vector engineering HALP!!

[Nico B.]

So I've downloaded vector 1302 for snap gene and have my gene of interest but missing the understanding of how to cut the plasmid with either CRISPR or GB 2.0 for my minipreps... I guess my questions are how to i choose my sites for cleavage and how do I choose the promoter/terminator for GB for insertion into my binary vector?

I'd love a simple step by step. my current understanding is such:
- GOI -> E. coli

- denature E. coli -> PCR

- ?
- Mini prep -> Agro via freeze/thaw

- plant transformation

GB has me reading on IIS restriction enzymes for binary assembly, though now that I'm playing with snapgene and have my destination vector I don't know what to do. HALP!!

Thanks,

Nico

[Koeng]

CRISPR is used for in vivo cutting. It's fairly difficult to use in vitro, meaning you *won't* be using that for classic cloning. GoldenBraid 2.0 takes a pretty large library of parts to actually use, and I guess you don't have those because they're not simply on addgene, you have to ask for them. (I've looked into GoldenBraid extensively because I am designing a golden gate - derived assembly method that will be more efficient)

In my opinion from what you've wrote, GoldenBraid isn't what you're going to want to be using. The vector you have isn't even compatible with GoldenBraid to begin with. 

I would recommend you use Gibson assembly. You can conveniently go over to Action menu in snapgene and click on the gibson assembly tab. That'll give you the tools needed to design primers for your gibson assembly. After you finish it, check out your file. If it checks out fine, go into history. It'll show you fragments with all the PCRs you did. Click to retrieve those files.

In them, the primers should be annealed onto the fragment with some overhang. Take *only* the part that is annealed and put the sequence into this tool- http://tmcalculator.neb.com/#!/ . This should show the annealing temperatures you'll need for your PCR, and it can make the reaction a lot more efficient. Personally, I would highly recommend Q5 polymerase for your PCRs. You gotta use a polymerase like it anyway for good gibsons, and you can add a sample of gibson mastermix to your order of Q5 ( https://www.neb.com/products/special-offers/nebuilder-hifi-dna-assembly ) for free, which is great. While you're at it, pick up some DpnI - https://www.neb.com/products/r0176-dpni . 

A note on the promoter/ terminator pairs: you have GFP currently expressed under the CaMV 35S promoter. When gibsoning, remove it and replace it with your CDS

What you'll wanna do next:

1: Miniprep the current plasmids you have.

You probably have pCAMBIA1302 along with another, so miniprep both of them from your E coli samples. You'll want fairly high quality DNA.

2: PCR off of the miniprep. 

Follow the protocol here- https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491 . Since you might not have something to quantify the DNA, just add in 1µl of it. This would normally not be recommended, but I get through that later. You'll want to do a 50µl PCR reaction.

3. Run a gel on your PCR. 

Only run 10µl of your PCR reaction. Just a normal electrophoresis run, it will be to make sure your PCR's worked. Just follow this https://www.addgene.org/plasmid-protocols/gel-electrophoresis/ if you want to get a bit more familiar with it. After seeing that your PCR worked, go onto the next step

4. DpnI digest your PCR product

Directly add 1µl of DpnI to the 40µl of your PCR reaction. This can also be done while you're doing your gel, making things go along much quicker. (It works in PCR buffer. https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-a-q5-taq-or-phusion-pcr-mix , plus that's what I do and I can personally vouch it works). This step is remove all the original miniprepped plasmid from your reaction. Why not just gel purify? A lot of times UV can degrade DNA which lowers reaction efficiency. After 1 hour at 37c and 20 minutes at 80c, take the reaction out and purify it

5. Purify the reaction. If you don't have a cleanup kit, just ask mobio for a sample- http://www.mobio.com/samples-all/ultraclean-15-dna-purification-kit-sample.html . It's a good kit, though it takes a bit longer than the kits I usually use from zymo. Both I recommend.

6. Gibson assembly. If you have 1 insert, put 7.5µl of that and 2.5µl of PCRed vector along with 10µl of HiFi mastermix (Note: It is assumed you don't have anything to quantify DNA. I know this is unoptimized reaction, but it will still work). Put that at 50c for 1 hour.

7. Transform. If you plan on making your own competent cells, don't. Go ask lucigen for a sample: http://lucigen.com/request_sample.html . The chemically competent cells work great, and I have often used them when I really don't want to make competent cells myself. The customer service is also very good there, which I appreciate a lot. 

8. Plate. Self explanatory. I prefer to transform onto 2 plates, one where I streak and one where I use beads. Of course, this is pretty wasteful, but it makes my day a lot better to see a nice even amount of colonies on my plates while still being able to pick them individually. 

9. Pick 3 colonies and liquid culture

10. Miniprep those and sequence. I use genewiz for my personal sequencing, they give good results. When you miniprep your cells, you'll want to elute in 35µl. Send 10µl to sequence with them, with your primer in whatever concentration they ask for.

11. Win. Get a sequence verified plasmid that you want! 

This is for the cloning portion. Sebastian or another plant scientist can help more with the Agro/plant part, I only work with yeast and E coli. Hope this helps with your cloning. Btw, if it's not confidential you might want to post what DNA you want to specifically clone.

-Koeng

(DISCLAIMER: I am not associated with any companies mentioned, I just find their products nice plus most have free stuff which is an added bonus. Also, this is how I clone things when using gibson assembly, which I personally believe is the best assembly method for new biologists. This is my opinion based off of experience with cloning. GoldenGate is a nice assembly method for *modularity*, not for speed or simplicity, which is what gibson excels at. GoldenBraid, using it's binary expansion-GoldenGate, feels like the offspring of MoClo and Biobricks. While I'd agree it's great for a lab with the system setup, it's not too great for sharing parts. Unlike Biobrick, everything is not compatible with everything else, which to me is a big turn-off. I should be able to reassign part1 to be in front of part2 or vice versa or reverse them while doing that, in a single reaction, with them all in the same vector. Biobricks take forever to clone, and MoClo has too many vectors and too few ways to manipulate parts in those vectors. No wonder why a lot of labs use gibson assembly to put things into goldengate vectors. Actually, the only labs I know that use goldengate methods and derivatives (moclo/goldenbraid) do it simply for variant libraries. Too much 'activation energy' needed to get the system running in a new lab without a need for those libraries. Anyways, enough ranting on cloning)

[Mega [Andreas Stuermer]]

Cutting the plasmid on the computer or in real?

IRL, you have 10'000'000'000 copies of that plasmid dissolved in 1mL water. You take 20 uL from it and add an enzyme like BglII. It then cuts the DNA at the given sites.

If your insert was also cut with BglII it has compatible overhangs and you can ligate