Hi.My name is Mark Blo. I'm a graduate student of biology at the University of Mines in Germany. Today, my friend Kajo and me would like to show you how to perform a homemade site directed neurogenesis.
The method was originally developed by Ethal and was adapted by many others. So when you look on the internet today, we'll find plenty of different protocols with which differ in some more or less significant points. Today, we would like to show you a simple and cost efficient way that works for us well.
So let's get started. For this experiment, you need a thermo stable proofreading, DNA polymerase. We always use PFU polymerase from fermenters.Logically.
You also need the polymerase buffer, DNTP, mix and water. Further, you need the restriction enzyme, DPN one, competent e coli cells, an auger plate, bacterial culture broth, and a bunch of toothpicks. The most essential and critical part of this method is the design of the mutagenic primers.
There are a few simple guidelines you must consider when designing your primer pair. The primers should be complimentary to each other and between 25 and 45 nucleotides in length. The mutations are inserted in the form of mismatches to the original plasmid.
The mismatches must be contained in both primers and should lie in the middle of the primer, flanked by at least eight nucleotides on each side. The primers should have a GC content of at least 40%and have one or more Gs and Cs in their five prime and three prime ends. Prior to using PCR, primers should be desalted phosphorylation.
FPLC or page purification is not necessary. There is no need to calculate the melting temperature. Simply make sure that the melting temperature on the shipping certificate is higher than 60 degrees C.For screening purposes, we always add or delete a restriction site from the plasmid.
For details on that, please check our written protocol. The first step in the whole procedure is setting up the thermocycling reaction. For this, you need 10 to 60 nanograms of the plasmid you want to mutate.
It is critical that the plasmid was isolated from a damp positive bacterial strain. Then add about 150 nanograms of every mutagenic primer. 1.5 microliter of a one in 10 diluted a hundred Picomolar stock will give 150 nanograms of the primer.
Complete the reaction with dn, tps, polymerase buffer, PFU polymerase and water. We set the initial and recurring de maturation time and temperature on a thermocycler to 30 seconds. At 95 degrees C, the ealing temperature and time is set to 55 degrees C and one minute.
The polymerase. We use calls for an elongation temperature of 72 degrees C.To decipher elongation time. We calculate one minute per kilo base of plasmid and add an extra minute.
To that time, 18 cycles are sufficient to create enough mutated plasmid directly after the cycling has finished load five microliter of the reaction onto a 1%TAE agros gel. If the amplification was successful, you should see a distinct band to eliminate the original plasmid from the reaction. We add one microliter of the methylation sensitive restriction enzyme DPN one.
Then we incubate the restriction digestion for at least one hour at 37 degrees C.After that, five microliter from the DPN one digestion are transformed into competent cells. For this purpose, we always use standard heat shock transformation. The competent cells are a key factor which determines the successfulness of this method.
Therefore, moderate or highly competent cells are preferable. After one night of incubation colonies should appear because the mutation efficiency is not a hundred percent. We need to screen for the mutants.
We do this by restriction digestion, so on day two of the procedure, we only need to select some clones and grow them overnight for mini prep on the next day. On the next day after the mini prep, we perform a restriction digestion with the marker restriction enzyme. We then load the restriction digestions onto an agro agros gel, and perform an electrophoresis.
On the finished gel, you can directly see which of your colonies carries the successfully mutated plasmid. In this case, we deleted an eco Armon site while inserting the mutation. So the third band from the left represents a negative clone while all the others are positive.
Nevertheless, you should confirm your successful mutation by sequencing. That already was it. As we said, this is a very simple and straightforward method.
Without any great pitfalls, we hope that our tutorial will help you to save lots of costs and increase the throughput in your lab. We wish you good luck for your research. Thanks for watching.
