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Any biologists/genetic engineers?

Anonymous Mon 08 Mar 23:27:20 2021 No.12808534 View Reply Original Report

Quoted By: >>12809800 >>12809821 >>12810118 >>12810880

Question: I'm looking at expressing some protein in bacteria by using a plasmid as the vector The protein has a known corresponding DNA sequence.

I'm using snapgene to try and design the plasmid. I keep hearing over and over about the importance of cloning sites. Which would allow for the ligation of the desired DNA seq into said plasmid.

Why is this at all necessary? Snapgene for example allows me to simply cut and paste my desired DNA into an existing plasmid. And it seems I can order it just like that. So why would anyone mess around with cloning sites and an extra ligation step when they can just have their target gene synthesized directly into the plasmid from the start? I must be missing something

Anonymous

Anonymous Tue 09 Mar 2021 06:18:45 No.12809800 Report

Quoted By: >>12810774

>>12808534
>have their target gene synthesized directly into the plasmid
>can order it just like that
How do you think that synthesis would be done without using cloning sites and ligation? Practical oligo synthesis lengths are short, the plasmid factory will still need cloning sites and ligation to make it. And you choosing which ligation sites flank your gene means you can cut and paste other sequences your lab has later instead of ordering an entire new plasmid.

Anonymous

Anonymous Tue 09 Mar 2021 06:30:12 No.12809821 Report

Quoted By: >>12810774

>>12808534
A) its more expensive to order the custom insertion than to get an undergrad or phd student to do it at slave wages
B) you still need your insert to fit in the RE sites because the lab techs need to stick the insert into the plasmid

Anonymous

Anonymous Tue 09 Mar 2021 08:24:58 No.12810118 Report

Quoted By: >>12810774

>>12808534
Full plasmid cloning do exists and are used quite extensively but the oligos you will make are going to be rather "bad" (long and high melting temperatures consequently) as well as you probably will not be able to reuse them when you will want to insert the same gene into another plasmid.

Anonymous

Anonymous Tue 09 Mar 2021 12:52:09 No.12810774 Report

Quoted By: >>12810880

>>12809800
>>12809821
>>12810118
I think I understand.

What I'm trying to do is take an operon that exists in a massive cosmid packed full of other useless shit, and express that operon in a bacteria.

So, it sounds like the best way is to order the cosmid containing the gene of interest, find an expression vector with matching RE sites, and then perform restriction digest on the two together?

Anonymous

Anonymous Tue 09 Mar 2021 13:42:08 No.12810880 Report

Quoted By: >>12810893 >>12810935

>>12808534
>Why is this at all necessary? Snapgene for example allows me to simply cut and paste my desired DNA into an existing plasmid. And it seems I can order it just like that. So why would anyone mess around with cloning sites and an extra ligation step when they can just have their target gene synthesized directly into the plasmid from the start?
As others have mentioned, its "free" to the PI to have the undergrads/grads do it. Ordering is expensive as fuck. 9/10 times I have the vector I want and the insert lying around in the lab, so I'll just spend a week cloning something.
That said, multiple cloning sites are only needed for restriction enzyme cloning, newer strategies like Gibson assembly are the de facto and don't require any cloning sites. Lots of PIs refuse to move on from restrictions enzyme cloning because "we have all these enzymes in the freeeeezer" sunk cost fallacy. It's kinda infuriating.
>>12810774
First, ordering DNA will require some amount of proof that you work for a lab, they (generally) wont ship stuff to personal addresses and will certainly flag/watch you carefully if they do allow it.
If you work for a lab- see if what you want is on addgene and order it for $50. Or order the insert + something with the vector of your choice and clone from there.
FYI, if you've never done cloning, and don't have the materials- you won't be able to do shit. The cloning materials alone are worth more than half a grand (not including the needed equipment, which will require several grand investment) and require special storage for something like restriction enzymes (-80 in a no-thaw freezer, on ice when in use), selection markers + competent bacteria + their storage/growth media (you DO have competent bacteria, don't you?), and even with all the lab equipment, cloning is a bitch and just doesn't work 50% of the time.
Oh, and you'll have to mini-prep + send out for sequencing to make sure your cloning worked and you don't have any indels.

Anonymous

Anonymous Tue 09 Mar 2021 13:50:25 No.12810893 Report

Quoted By: >>12810935

>>12810880
>(-80 in a no-thaw freezer, on ice when in use)
-20, not -80; the -80 is for the competent bacteria storage.
Regardless, all lab equipment + supplies will not ship to home addresses, even if the person ordering it is from a lab.
And you best believe the government will be interested in what someone is trying to order plasmids/DNA of any sort and you will get a knock on the door if its even REMOTELY a sketchy insert (even if its not, I wouldn't be surprised).
So don't try.

Anonymous

Anonymous Tue 09 Mar 2021 14:01:53 No.12810935 Report

Quoted By: >>12810964 >>12810969

>>12810880
>>12810893
Alright thanks. Yeah I'm actually at a community college but we have electroporation setup and the other stuff.

The gene operon I want is pretty obscure and doesn't seem available anywhere on its own. So I will have to order the native plasmid + the expression plasmid and recombine them, right?

Or can I just have the insert synthesized? I have the full nucleotide sequence

Anonymous

Anonymous Tue 09 Mar 2021 14:10:55 No.12810964 Report

Quoted By: >>12810969 >>12810997

>>12810935
>So I will have to order the native plasmid + the expression plasmid and recombine them, right?
>Or can I just have the insert synthesized? I have the full nucleotide sequence
As long as this is a college project-
If the sequence isn't too long, you could easily have it synthesized. If you are doing this with restriction enzyme cloning, you would need to synthesize it with the restriction sites added on to the insert on either end. So say you have a multiple-cloning site in your vector, and decide to pick EcoR1 and Sal1, for example, you would then synthesize Ecor1+insert+Sal1. That will allow you to digest the insert and get the cut ends which can be ligated to the cut vector.
https://www.addgene.org/protocols/subcloning/ is good to check out for the whole process.

Anonymous

Anonymous Tue 09 Mar 2021 14:12:52 No.12810969 Report

Quoted By: >>12810997

>>12810964
>>12810935
But check prices. If the native plasmid is available and cheaper, just order that. You can basically get your insert/vector any way you want, but make sure it has matching restriction sites.
Or if you want, you can order the insert and then PCR it up using primers with the restriction sites added on (which is what is usually done if you are cloning an insert from another vector). There's a few ways to do it, as long as you have 1) vector with cut restriction sites, 2) insert with matching restriction ends

Anonymous

Anonymous Tue 09 Mar 2021 14:24:44 No.12810997 Report

Quoted By:

>>12810964
>>12810969
the operon is about 7000 base pairs long. If that's within the acceptable range for a synthetic insert, then it sounds easier, no? Otherwise I'd have to fish around for an expression plasmid with RE sites which match whatever there is in the native plasmid, which will probably contain extraneous crap, right? Whereas the synthetic DNA I can add any RE sites to fit my expression vector of choice. I might also look at adding some kind of selection marker to that insert.
appreciate the help btw

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