Hi, While I do not think that the comments (at least those posted to the board) were evil or derogatory, I nevertheless would like to emphasize the need for details.
With appropriate cloning strategy artefacts such as those described by Jan could be avoided almost entirely. While 'in silico' cloning may be helpful, consideration of major factors that can affect the process is much more important. In silico stuff can be a bit misleading - for an example I highly recommend interested parties to check out Anton Yuryev's book 'PCR primer design'; specifically chapter 3 which discusses several common myths regarding PCR primer design (it turns out that casually designed PCR primers only work in ~70-75% of cases). Casual use of programs does not make things better. Overall, there are too many unknown branch points in a 'straightforward' process such as cloning and without details the diagnosis of a problem becomes equivalent to leaving your car mechanic a phone message 'my car does not start, tell me how I can fix it'. Undoubtedly the mechanic would want to know a little more about the problem at hand before suggesting an answer. It is didactic to read about cases like this one, and this list is being read by many - including both molecular biology professionals as well as novices. So, details are useful and not just to the person who asked the question. For example - if no colonies form after ligation, assuming that all the basics have been checked (such as, properly designed amplification primer sequences, compatible sticky ends, compatible enzymes, & suitable restriction buffers to name just a few) the typical causes that should be investigated further are: 0. Bad/incorrect/old/warmed-up competent cells. Imagine a freezer dying briefly in the night, then recovering without anyone knowing. If you're lucky your freezers are alarmed and chart-recorded. Yet even this still does not prevent another user from carelessly taking competent cells out for too long, not noticing, or failing to replace the compromised stock. 1. Too much insert - resulting in 1:2 ligation with incompatible sticky ends. 2. Toxic insert - which is why the e.g. T7 promoter is oh so good since cloning-grade cells do not have the right polymerase for it. 3. Star activity or unexpected cleavage site (i.e. the genetic database tells you one thing but your source DNA is from a mutant). 4. Old/bad enzymes (almost does not happen these days although some brands of DNA ligase expire faster than others). 5. Contaminated enzymes (happens more than we'd all like to see in labs with multiple users of the same reagents). 6. Wrong/contaminated antibiotics (see previous comment regarding multiple users) 7. mis-synthesized primers. Just a couple of weeks ago we had this happen - a desperate sequencing attempt (of a third-step intermediate) confirmed an error in one of the primers we used to produce a complicated construct that kept failing time and time again. And that's just a few options, and only for one specific case - 'no colonies after ligation, transformation, and plating'. On a much more positive note, the overall process works. It has been developed over decades, with continuous improvement by many extremely talented people. But don't let the apparent simplicity fool you - there are pitfalls present at every level. Still, it works more times than it does not - and it's a whole lot easier than it used to be (cesium chloride gradients, anyone?). We just had an intern (undergraduate student) who came in with basic lab skills and had no prior experience in molecular biology, crystallography, etc. but was able to clone, express, purify, and crystallize a novel (soluble bacterial) protein. We solved the structure before his internship ended. So, overall the process clearly works :) Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sridhar Prasad Sent: Sunday, August 31, 2008 12:28 AM To: [email protected] Subject: Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff I want to draw attention to some of the early unruly response(s) to this question. First of all, no one is forcing anyone to respond to each and every message that is posted on the CCP4 bulletin board. If you are so keen on responding, please be respectful and considerate and think twice before you click on the send icon on your computer with derogatory remarks. Thanks Sridhar Prasad -----Original Message----- From: CCP4 bulletin board on behalf of Jan Schoepe Sent: Sat 8/30/2008 8:52 PM To: [email protected] Subject: [ccp4bb] AW: [ccp4bb] hi / cloning stuff Hi, I think your question is quite reasonable no matter what others say about it. There were even people asking about how to make buffers and then a big discussion about the HH equation arose. And we should not forget that modern crystallization starts with cloning ;-) Anyway, you should have given us some more details about your experiments (cells, vectors etc.) and maybe a better subject. If you want to clone "something", it is a good idea to do it in silico (e.g. with Clone Manager or Vector NTI) before you walk into a wet lab to check, that things can work as desired or if there are fishy things like internal restriction sides etc. Let me tell you a story from my PhD thesis, maybe this will help you somehow. I started with cloning 10 genes into a before modified pET vector (for E. coli overexpression). After cleavage of the vector I got cohesive ends ("sticky ends"). The genes were PCR amplified from the chromosome of another bacterium. So things should have been very straight forward. Sticky ends should make sure that the amplified genes were inserted in the right direction and the cleaved vector should not ligate without the insert (and of course there was antibiotic selection). But what I observed was often quite different from that. Often agar plates stayed empty after transformation. That means, the cells were not able to grow, because there was no vector in the cells that made them be able to survive on the antibiotic plates. When I got colonies, the DNA gel looked very strange (not possible to interpret) or the vector closed without the insert. In this case, E. coli seems to be able to "repair" the cohesive ends. Finally, I got it done but it took me months. In one case, a plate seemed to be empty but I did not wanted to throw it away so I let it stay a litte longer (~another day?, I do not remeber exactly, but it was for a long time...) at 37° C than just over night (~0.5-1 days). Finally, one single colony showed up and it was one of the right ones. With another vector it might be possible to do the same thing within weeks. This is just cloning luck. Another story, even if it could be embarassing to mention it here. In one case, I got an Xtal structure but then I wanted to do some more activity tests. I found out that my glycerol stocks were not alright any more and I had no plasmid backups (do it!!!). So I had to clone, again. But in this case I used a cloning kit from Qiagen (I have no commercial interests here). I got the insert into the cloning vector from Qiagen very easily. Then I was able to amplify it as much as I wanted and the ligation procedure was not so dependent from the yield of the PCR reaction (high molar access of insert compared to the cleaved vector) . Also in this case, cloning into the expresson vector was not completely trivial (maybe 1-1.5 months) but much easier than without this kit. So, it might be an idea to use a kit that you can amplify your insert as much as you want. I know that these kits a pricy but your time is also expensive and you also use/waste a lot of chemicals for unsuccessful trials. After I finished my thesis, another PhD student started to work with these vectors and tried it for more than one year and then gave it up! So this vector really seems to be a tough one. Thats why if nothing works, you might take another vector into consideration. But before that you should check buffers, ligase, restriction enzymes etc. and do in silico cloning. Another idea would be do to de/phosphorylation (see textbooks). Good luck! Jan --- vijay srivastava <[EMAIL PROTECTED]> schrieb am Sa, 30.8.2008: Von: vijay srivastava <[EMAIL PROTECTED]> Betreff: [ccp4bb] hi An: [email protected] Datum: Samstag, 30. August 2008, 13:04 hi i am facing problem in cloning,getting my insert and vector at the correct position after digestion but after ligation colony is not coming and if how it is coming than i am not getting my insert Unlimited freedom, unlimited storage. Get it now __________________________________________________ Do You Yahoo!? Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden Schutz gegen Massenmails. http://mail.yahoo.com
