Loki I love the progress reports, this is gonna be costly just due to the amounts of materials used. Why the change in plans fro pre-made primers?

Yeah, its not going to be cheap, but so far, I think I'm going to be under $2000 for everything.
That's if there aren't anymore changes in the plans, major changes.

The change from premade primers is simply because I felt the need to post the primers was important.
The primers are the key to the whole process really, without them, your results will vary greatly.
So you could do everything exactly the same, but with slightly different primers
and things might work, but they might not...

Posting the primer sequences presents a problem though.
The primer sequences are unique, so LEO would only have to email the sequences
to the places that make primers, and I'd be the only one, ever, to order those primers... >busted<

Of course, there's still a chance even with the same primers that they might not work
on DNA from another strain of marijuana, but that chance is extremely small...
But making my own primers will have the side benefit of allowing me to create primers
for Cannaboid genes that I don't know the sequences of, but can try and guess...

-Loki

Heh, I've never been quoted before...
Closest was a sort of tale involving the fact
that the only Spanish I know is "Give me the gun."

But that's another thread...

-Loki

Well, the Phleomycin resistant gene is being expressed in both cultures, but not Ampicillin resistance.

Which means, the second gene isn't being expressed.

I didn't use the Cre-Lox Recombination tehnique, so there were 12 base pairs between the two genes.
I was hoping the extra DNA between the genes was small enough, so it wouldn't matter...

Another thing might be the stop codon after the first gene. There are 3 possible stop codons.
I thought the one I used would have marked the end of the first gene and then let the next gene be expressed.

I'll have to try again with the other two stop codons,
and use the Cre-Lox Recombination technique to get rid of the 12 base pairs between the genes.

-Loki

I was a little surprised that no one has objected to genetically engineered marijuana...

Don't worry... There won't be a repeat of GMO Cow Incident...


___________ MOOOOOOOOOOOOOOOOOOOOOO!!!!!!!!


-Loki

as long as the plants do not come out looking like the picture you displayed, what is there to object about?

even if there is objection - fuck it

is it going to stop you?

i certainly hope not

i think it is great to be able to observe the workings in a biological program, generally speaking

i can confidently say this is some ground-breaking stuff and the ground will give-way eventually

I'll try to keep the killer mutants to a minimum...



-Loki

My friend the genetics guy has a response;
So I work in a genetics lab and I've got a few questions/comments about this... not trying to be a downer, but here we go:
1. plants don't have plasmids, so you can't just mix E. coli plasmids with plant plasmids, because one doesn't exist
2. That't not how you engineer a plant. With that size of a piece of DNA you would probably want to build an artificial chromosome. So you're not going to cross bacteria with anything really.
3. you can't just smack a promoter onto one end of the DNA segment with so many genes running off of it. You're treating it like an operon, but that's more a bacteria thing. And you're not considering that you don't know what proteins are involved in protein folding. So you could move over all the genes sucessfully and you still might not get anything. But if you try to put in everything as an operon like that you're not going to get anything probably.
4. With DNA segments that large you're probably not going to want to use PCR to amplify the DNA, except for the small fragments.
5. Designing primers, I'm not even sure what you're talking about because the hardest thing about designing primers is for one knowing where the gene ends and begins, which the bigger problem is knowing where the promoter ends and the gene begins, and the second is is your promoter sequence so common in the chromosome that you're going to accidently amplify other DNA segments that you don't want to?

Your desire seems strong enough, but I'm afraid your knowledge of genetics isn't good enough to ever really do this. You're talking about doing something very complex here, and you're talking about moving over multiple genes and you don't even really know how they work or how they are produced, folded, etc, and I don't think you realize just how impossible it is going to be for you to do this.

Wrong. "Plasmids can be found in all three major kingdoms, Archea, Bacteria and Eukaryote" - Wikipedia
(There was a typo back a few posts where I mention plant plasmids, but I ment to say protoplasts...
but you are correct, a plasmid in a plant cell wouldn't be expressed,
so it wouldn't work.)


The bacteria is just the vector. I don't intend to cross bacteria genes with anything...
The protocol I plan to used is from here...

Plant Tissue Culture Concepts and Laboratory Exercises Second Edition


If that doesn't work. I'll use the same E Coli cultures
and remove the DNA with a plasmid kit and transfect with something like QIAconnect.
But I'll try the other method first, as I have the materials already.
I know its not the most effective method, but it does work or could work.

There are two protocols I'm going to try. One is E Coli carrying target plasmids
mixed with African Violet protoplasts (almost said plasmids again) and restriction enzymes and heat shocked.

The other protocol is basically, take the DNA from the E Coli, add it to the African Violet protoplasts,
15 minutes in a 42 degree sonic water bath, and plate.
Then cross your fingers and hope some DNA giggled in...
But if those don't work, and they may not, I can always fall back on a transfection kit.


While this appears to be the case, I've seen research that indicates otherwise.
Maybe I am thinking of bacteria, but I could swear the paper I saw on it was Eukaryotes.
It discussed controlling expression not only at the DNA level, but at the transcription and translation phases.
This is the promoter sequence I'm going to use.


Maybe, but maybe not...


What would you suggest then? I'm using PCR to isolate and assemble the target DNA,
the E Coli will amplify the DNA I use to transfect the African Violet protoplasts.


Well, we're even here, because I don't know what you're saying either.
But you forgot you also have to make sure the primers don't hair-pin or self-dimer...
I know my promoter sequence and the gene sequences.
I can't really use primer-BLAST, but I can use gels
to make sure any unwanted fragments are left behind.


Here we disagree again. My knowledge isn't good enough to ever really do this?
This is my first attempt. It may fail, but I will learn from it and try again.
But won't your "genetics" buddy look like a retard if I do succeed? I've only started a few months ago.
None of this is really that out of the box, especially transfecting African Violets.
All the tools and information is out there.


I understand its complex, but its not impossible.
If I have to step up my project to the next level, when PCR and Gels are no longer enough, I will.
I've already begun to looking into setting up a front company to get the FDA certification
to get the equipment I might need. I can get a used DNA sequencer on eBay for 2-5 grand...

-Loki

I have to admit that I am impressed with the way you handled the criticism. It's been a while since I have seen someone take it on the chin like that and remain cool. The odd bit of comedy "But won't your "genetic" buddy look like a retard if I do succeed?" makes it worth the read too! I almost blew Coke out of my nose when I read that. Keep up the good work Loki. I am behind you all the way! Unless of course you are wrong.... then I was just humoring you. =)