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In Topic: Explanation of "In-Frame" cloning?
22 October 2012 - 11:31 AM
Thanks! I think I fully understand now
In Topic: Explanation of "In-Frame" cloning?
21 October 2012 - 10:40 AM
Oh eff. I didn't even think about the orientation of the blunt end ligation. Thanks a bunch!
Maybe I'm taking the part about the methionine (ATG), but I don't think I really understand the issue? The n-terminus should start with the fluorescent tag so it needs to have the start codon, ATG. Translation is going to keep running through to the end of my gene of interest since there's no stop codon on my flourescent protein. The original start codon for my gene of interest wouldn't matter would it since there would be methionines in the internal sequence of the protein?
So I just need to emphasize this question one more time because I don't want to go through rounds of cloning and then do a final transformation and find that it doesn't work, haha (I need to transform into B. subtilis and it takes an entire day
).
[Assuming that my product goes in correctly (correct orientation) via blunt end ligation]. When I digest the topo vector to retrieve my insert and my 'next vector' with the same restriction enzyme and ligate those two together, will that ensure that my insert goes in in-frame?
Maybe I'm taking the part about the methionine (ATG), but I don't think I really understand the issue? The n-terminus should start with the fluorescent tag so it needs to have the start codon, ATG. Translation is going to keep running through to the end of my gene of interest since there's no stop codon on my flourescent protein. The original start codon for my gene of interest wouldn't matter would it since there would be methionines in the internal sequence of the protein?
So I just need to emphasize this question one more time because I don't want to go through rounds of cloning and then do a final transformation and find that it doesn't work, haha (I need to transform into B. subtilis and it takes an entire day
).[Assuming that my product goes in correctly (correct orientation) via blunt end ligation]. When I digest the topo vector to retrieve my insert and my 'next vector' with the same restriction enzyme and ligate those two together, will that ensure that my insert goes in in-frame?
In Topic: Explanation of "In-Frame" cloning?
20 October 2012 - 07:38 PM
I'm actually putting a flourescent tag on my protein so my tag will have the start codon, and continue to run all the way to the end of my insert where the stop codon is.
Let's say my tag and gene are shown below, respectively.
5'- ATG CTG GTG ... AGA AGT TGT - 3'; 5'- ATG GGC GTG ... GGG TTT TAG
I'm going to design my primer such that it will hybrize the two genes together in later PCRs, so I put an overhang on my tag's reverse primer that is complementary to the the beginning of the desired insert's sequence:
(Tag Reverse primer)
5'- CAC CGC CAT ACA ACT TCT - 3'
I'm putting it into the TOPO zero blunt end vector, which I don't think is going to be a problem in terms of in-frame (if I understand it correctly) since there's a topoisomerase that just sorta unwinds my PCR strands in. Here's the map of the vector (page 18): http://tools.invitro...unttopo_man.pdf
My question is that, after I put it into the TOPO vector, which I'm pretty sure will go 'in frame', can I digest the TOPO vector with ECORI since it flanks my insert, and place that into another vector that has a multiple cloning site for ECORI.
Thanks for your quick reply, by the way
Let's say my tag and gene are shown below, respectively.
5'- ATG CTG GTG ... AGA AGT TGT - 3'; 5'- ATG GGC GTG ... GGG TTT TAG
I'm going to design my primer such that it will hybrize the two genes together in later PCRs, so I put an overhang on my tag's reverse primer that is complementary to the the beginning of the desired insert's sequence:
(Tag Reverse primer)
5'- CAC CGC CAT ACA ACT TCT - 3'
I'm putting it into the TOPO zero blunt end vector, which I don't think is going to be a problem in terms of in-frame (if I understand it correctly) since there's a topoisomerase that just sorta unwinds my PCR strands in. Here's the map of the vector (page 18): http://tools.invitro...unttopo_man.pdf
My question is that, after I put it into the TOPO vector, which I'm pretty sure will go 'in frame', can I digest the TOPO vector with ECORI since it flanks my insert, and place that into another vector that has a multiple cloning site for ECORI.
Thanks for your quick reply, by the way
