tag:blogger.com,1999:blog-7497105671108206262.post1909206240213072968..comments2023-10-28T07:51:34.506-07:00Comments on MolBio Research Highlights: An alternative cloning strategy: yeast recombinational cloningAlejandro Montenegro-Monterohttp://www.blogger.com/profile/18078462764857337905noreply@blogger.comBlogger9125tag:blogger.com,1999:blog-7497105671108206262.post-41819124942388405202010-03-25T14:18:11.757-07:002010-03-25T14:18:11.757-07:00Is there a limit on the number of amplicons you ca...<i>Is there a limit on the number of amplicons you can put together this way?</i><br /><br />We've tried 8 fragments (up to 1kb in size) with no problems. We haven't tried more or longer fragments, so I couldn't tell you what the limit is.Alejandro Montenegro-Monterohttps://www.blogger.com/profile/18078462764857337905noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-90754942436570213322010-03-25T13:58:33.930-07:002010-03-25T13:58:33.930-07:00Well, for the procedure Nikolai explained I think ...Well, for the procedure Nikolai explained I think that the costs involved may be limiting for many labs. The other minor thing I´d like to comment is that fusion pcr is a very complex technique (in my hands I could only get it to work for up to 4 kbs). <br />In the case you want to build several constructs (or scaling up to 96 wells), the amount of primers needed for each particular reaction, Francisco Barrigahttps://www.blogger.com/profile/04331796971961157992noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-11882171577983450042010-03-25T13:37:25.279-07:002010-03-25T13:37:25.279-07:00Hi Nikolai,
Electroporation cuvettes are more exp...Hi Nikolai,<br /><br />Electroporation cuvettes are more expensive than making a CaCl2 solution :-). In Chile, grants are smaller and reagents cost 3 times more.<br /><br /><i>Are you sure that in vitro assembly would be more difficult?</i><br /><br />It's not about being more "difficult" technically, but it will require the purchase of more primers and you'll be adding another Alejandro Montenegro-Monterohttps://www.blogger.com/profile/18078462764857337905noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-43203202632830266002010-03-25T13:33:28.340-07:002010-03-25T13:33:28.340-07:00Though I just looked through the Colot 2006 paper ...Though I just looked through the Colot 2006 paper and realized that recombination will do the assembly for you in a single in vivo step, which makes the in vitro reaction I talked about pointless. Is there a limit on the number of amplicons you can put together this way.Unknownhttps://www.blogger.com/profile/12908331690423349420noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-88226593552251246412010-03-25T13:15:08.877-07:002010-03-25T13:15:08.877-07:00Electroporation is cheap if you make your own cell...Electroporation is cheap if you make your own cells. Just buy a small stock of your favorite electroporation strain (I like NEB's 5-alpha), grow them up, and prep as much as you need. There are some decent protocols on <a href="http://openwetware.org/wiki/Electrocompetent_cells" rel="nofollow"> OpenWetWare </a>. It does take a full day though, and the later washes can be tricky, since the Unknownhttps://www.blogger.com/profile/12908331690423349420noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-80645708726847878082010-03-25T09:45:05.724-07:002010-03-25T09:45:05.724-07:00Hey Nicolai,
Thanks for sharing your thoughts. Wh...Hey Nicolai,<br /><br />Thanks for sharing your thoughts. While I agree with most of your argument, I have some comments:<br /><br />I think that the time frames you mention are slightly wishful thinking. In practice, many of them will take longer, but as a general ballpark it's fine. <br /><br />Also, I mentioned phenol cleanup, because I was trying to list the cheapest way possible to do Alejandro Montenegro-Monterohttps://www.blogger.com/profile/18078462764857337905noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-35600403224881115562010-03-25T08:07:44.756-07:002010-03-25T08:07:44.756-07:00Though I haven't tried YRC myself, I'm not...Though I haven't tried YRC myself, I'm not sure that traditional cloning is as difficult as you make it seem. Let's look at how long cloning takes:<br /><br />1) Amplification of region of interest: 1.5-2 hrs if using a highly processive enzyme like Phusion<br /><br />2) Cleanup of PCR reaction: 0.5-1 hr w/silica column-based kits (why are you still doing phenol/chlorophorm/ethanol Unknownhttps://www.blogger.com/profile/12908331690423349420noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-57979816670265059932009-12-03T06:13:34.392-08:002009-12-03T06:13:34.392-08:00Hi! Thanks you for reading such a lengthy post :-)...Hi! Thanks you for reading such a lengthy post :-)<br /><br />Regarding your comments...<br /><br />I didn't include the gel band extraction/purification step in the last part because it serves the same purpose for what I was trying to say: there is an extra purification step. You are certainly right, anyway. Lots of people do that nowadays.<br /><br /><i>In my particular case, yeast is not Alejandro Montenegro-Monterohttps://www.blogger.com/profile/18078462764857337905noreply@blogger.comtag:blogger.com,1999:blog-7497105671108206262.post-13173125577009762442009-12-02T23:47:54.673-08:002009-12-02T23:47:54.673-08:00What you described initially is quite accurate reg...What you described initially is quite accurate regarding the steps involved (although most people use gel band purification kits instead of phenol/chloroform extraction) I just have two things to point out:<br /><br />1. If you´re lab uses yeast then YRC is the most straightforward strategy to clone. In my particular case, yeast is not an option and we have to stick to "old school" Francisco Barrigahttps://www.blogger.com/profile/04331796971961157992noreply@blogger.com