Most common mistakes - What mistakes are made during cloning? (Apr/27/2011 )

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

You want to know "mistakes in cloning" or "problems encountered in cloning"? This two might mean different things.

Arnold on Wed Apr 27 13:50:38 2011 said:

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?

hematopoietry on Thu Apr 28 09:03:23 2011 said:

Thanks for your reply and information. Writing a FAQ is only the practical part of the Thesis. I will also do a literature study on the problems occur during cloning (for example on ligation and transformation). Before I start the literature study I have to know which problems there are. That's why I'm searching on fora. After this little on-line research I will categorize these problems. For every general question I will look on Pubmed for literature. Maybe the literature study will not add much value but the practical part (FAQ) will be linked on a commercial computer program which could be used for cloning. In the FAQ there will be information on how this program could prevent these problems. Next week I will verify my ideas with the coordinator of the Thesis.

As an answer on your last question. Yes indeed, I'm from Holland. Haha I guess you're also from Holland.

Arnold on Thu Apr 28 09:51:09 2011 said:

Well, there is not much literature on cloning problems (actually, there is not much literature on failed experiments AT ALL). If you want to do a literature study on cloning, the closest you get is evaluating strategies that improve cloning efficiency. There is lots of interesting reading in this field; strategies to improve ligation, techniques to make highly competent cells. This also is important to your subject; if you manage to improve the efficiency of a bad reaction, you might be able to obtain your construct. By studying cloning efficiency you will also find clues on failed cloning. For example when a graph shows T4 ligase activity vs two-valent ion concentration, the 0 activity point tells you at which concentration your ligation fails because of low ligase activity.

You will see lots of cloning problems on this forum, so I think this is a good place to start.

Noord-Holland / Kennemercollege Buhlerlaan toevallig?

hematopoietry on Thu Apr 28 11:10:49 2011 said:

Thanks for your help. Yes that was what I'm thinking off. Looking in Pubmed on articles where they are improving the current strategies. This is my third day after I started working for the Thesis. At the moment I'm looking at the problems and improving my knowledge on cloning. During my Bachelor most study objects where in the Immunology, Patho(physio)logy and Oncology. At the moment improving my knowledge on cloning is really important. After a week I will make a plan and formulate the research questions and let my coordinator look at it.

I live in Noord-Holland. At the moment I'm studying at Utrecht University. You studied at the Kennemercollege? I went to the Bonhoeffer College in Castricum. In which institute/research field are you working at the moment?

Arnold on Thu Apr 28 11:45:33 2011 said:

>> check pm

I could barely clone anything when I was trying a 1:10 Vector:Insert ratio. Then I raised the ratio to 1:20 1:50 1:100 but I still never got anything until I lowered it to 1:3.