Arnold, on 27 April 2011 - 05:50 AM, said:
Hello All,
I'm writing a Bachelor thesis on the problems that could occur during cloning. After a week searching for questions I want to make a FAQ for cloning. On this forum I read a lot of discussions on problems with ligation and some problems found during analysing. A lot of people asked questions but do not tell if there problems are solved because of the replies in the topic.
Are there any people who want to tell witch problems they found during their cloning experiments? And how they solve these problems.
I'm also searching for scientific articles about problems that occur during cloning.
Thanks a lot for helping me.
Arnold
Although cloning guides could theoretically be a big help, in practice you find that each reaction is unique, and there is no 'standard recipe' for fixing a broken clonation (besides that, most scientists need to make the mistakes themselves, stubborn as they be). There are some things you should keep in mind when cloning
-Good controls: wherever possible, verify your reaction. Cloning is one of the many procedures where you don't know what you get until the end. Check your SAP with an empty vector ligation, check the digestion of your vector by transforming unligated / and unsapped vector. Check your competent cells with accompanying pUC19 vector. If you're unsure of your insert, cut it and run it on gel
-Use competent cells of the highest competency whenever you do a difficult cloning reaction. While DH5a cells might be fine for retransformations or sticky ligations, when you are ligating very large blunt fragments using the highest competency (10E9) cells could mean the difference between 0 and 20 clones. (Note there are a number of higher efficiency competent cells available, I suggest you test each of these to see which works best for your construct, then make your own competent cells with the Inoue protocol.) Of the things I checked this had the highest impact on the number of transformants.
What I found useful when improving the efficiency of my blunt ligation was evaluating a number of different ligation and transformation conditions (like PEG vs HCC vs i/j switch ligation, Fragment:Vector ratio, total amount of DNA in the ligation mix, amount of PEG-6000, amount of ligase used, amount of ligation mix transformed). Certain graphs might show a decrease in transformant number per plasmid when adding more ligation mix, keep in mind that the total number of transformants could still be higher (and this is all that matters). There is a whole list of things I could tell you that mattered for my reactions, but like I said before every reaction is unique.
I'm afraid there isn't much literature on troubleshooting ligations and transformation. You could have a look at the website of the manufacturers of the materials involved; you will find a section on troubleshooting. Some labs keep cloning guides and troubleshooting sections on their website. There is a lot of information on improving ligation and transformation efficiency in peer-reviewed literature, though (mostly from the 80s and 90s). Just use pubmed and you'll be sure to find them.
I am a little worried about this guide though. At least my university wouldn't let me get away with writing a FAQ for my Bachelor Thesis. Perhaps a literature study on ligations and transformations would've been enough, but because of the little added value (most scientists know how to clone) I highly doubt it. I would verify this before you start. Anyway, I wish you all the best with writing this guide, you will learn a lot about cloning while doing so (best way of course still being: do it).
Another thing. When I saw your name I couldn't help but wonder: are you from Holland?
Edited by hematopoietry, 28 April 2011 - 01:09 AM.
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