Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol. We have found that it is the best protocol. This protocol may be particularly useful if you are finding that your transformations are not working or yiedling few colonies.
In nature what happens is shown on the following two videos:
Overview
To see this in a nice lab demonstration tutorial about how transformation procedure is used, and why, watch this:
Materials
The demonstration of our iGEM protocol realized in summer 2009 is shown below:
Competent cells (we use DH5α)
DNA (this is a sample)
Ice
42°C water bath
37°C incubator
SOC (check for contamination!!)
Petri dishes with LB agar and appropriate antibiotic
Procedure
1. Start thawing the competent cells on crushed ice (we find this cells in the -70°C fridge)
2. Add 200μl competent cells and 2 or 5μl (50ng) DNA on ice
3. Incubate the cells for 30 minutes on ice
4. Heat shock at 42°C for 90 seconds water bath (not shaker)
5. Incubate for 5 minutes on ice
6. Add 200μl SOC broth (but sometimes not)
7. Shaker 2 hours at 37°C
8. (Sometimes centrifuge for 10 minutes at 10000 rpm and a few supernatant take int he dumb and suspendation the pellet)
9. Plate usually 60μl of the transformation or we make distribution 20μl and 200μl Petri dishes with agar and the appropriate antibiotic(s) with the part number, plasmid and antibiotic resistance
10. Incubate the plate at 37°C for 12-14 hours
Notes & troubleshooting
If you think another video demonstration would be needed, please go on to the next video:
More details about the procedure, with excellent links cand resource material can be found in the Molecular Biology Online Notebook.
References
1. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed.,1.25-1.28. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, USA.
Today I would like to speak with you about liquid handling in the lab. Majority of our reactions are performed in liquids. From culturing of the cells to the specific enzymatic reactions performed, all are done in liquids. This is why we need an accurate and easy liquid handling device. We ususally perform liquid handling with pipettes.
So what is a pipette? I am sure almost everyone saw a pipette. A pipette is a device that aspirates liquids in order to transfer it from one vessel to the other. You can have a good description about general topics here.
You can have a very-very good introduction in the history of the modern molecular biology pipettes from a video by Lim Leng Hiong.
So, let’s see what “Freshbrainz” tell us about pipets:
But what kind of pipettes do we use?
The most basic pipette is a single use plastic pipette. It is not very accurate, but you can transfer liquids from one tube to a different one.
You can use it like this:
We have a simillar pipette, a glas pipette that we use less for liquid transfer, but for removal of liquids from tubes. Usually after centrifugation steps we have a pellet and a liquid supernatant. If we want to discard the supernatant in a carefull and accurate way we use these “Pasteur” pipets. More details about Louis Pasteur here and please see a video about his work here.
So here is one of our Pasteur type, glas pipettes:
And here is how we use a Pasteur pipette:
Of course the majority of our work is done with the so called Gilson pipettes. As our friend Lim Leng Hiong explained you these were specially designed for molecular biology work.
Below is video you can see how we handle liquids with a Gilson pipette. Please pay attention to the two stops made with my thumb. The first stop is reached when we aspirate the desired volume, while the second stop when we dispense the liquid. There is a button which is used to remove the single use tip of the pipette. So, please watch carefully the demonstration:
We have traditionally three type of pipette tips and these are differntiated by their color.
The smallest volumes can be measured with the 2 ul (2 microliter) pipette. This pipette is considered accurate between 0.5 and 2 ul-s. The same tip is used for the 10ul pipette. We use this for volumes between 2ul-s and 10 ul-s. These pipettes are marked with gray, as shown below.
The tip used with this pipetes is here:
The next type of tip has yellow color traditionaly so the pipetes are marked with yellow:
The same rule: P20 should be used between 10-20uls P100 between 20-100uls and P200 between 100 and 200uls.
The same tip can be used for these three Gilson pipetes, namelly these ones:
The third type of this pipete is traditionally marked with blue. This is the one ml pipete. We call it P1000 and use it between 200 and 1000uls. Below is the head and the tip used for it.
With this set of pipettes you can perfom majority of molecular biology reactions in the lab in an accurate way. They are not cheap, the price of one pipete is in the range of hundered dollars. They are precision instruments, so usually each researcher has his own set to use. Please pay attention to this and never use someone else’s pipete set only she or he specifically alowed it to you.
You can have a look on the usage of these pipettes on the best tutorial I have ever seen, produced by the University of Leicester here:
OK but what other alternatives so we have?
We have two very usefull type of additional pipettes. One is called the multichanell pipete, you saw it on Lim Leng Hiong’s video, and the other is the repeater pipete.
The multichanel pipet we use can have 12 or 8 chanells and you can have a look on it here:
The range of volumes you can dispense with it can be seen here:
With these pipettes the volumes can dispensed can be adjusted in steps and not in a linear way. You can see the adjustment volumes for both type of multichanell pipettes here:
And here you have two videos about their usage:
The second type of very important help in the lab is the so called repeater pipete.
This pipete is able to dispense the same volume from a reservoir in a serial way.
Here is how it looks like:
The good stuff about these pipettes is that it can be used with different type of tips and it automatically recognizes the type of the tip you are using.
Here are the tips we use in general:
You can see on the next figure, that depending on the tip used the pipete is showing eighter 20 or 100 uls in the same position 1.
Here is a short video about how to use it:
With these pipets you can work easily in the lab. The master, the queen of lab pipettes is for sure the pipeting robot. We use a Tecan Genesis for pipeting smal volumes (5uls) in a serial way (e.g on a 384 well plate).
Have a look on this pipeting device:
In my next post I will come up with serological pipettes and the price of the water in the lab!
Stay tuned, and lat me know if you have any questions!
Labtutorials in Biology 