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J. Christopher Anderson
Приєднався 28 сер 2012
These videos are components of the online learning site www.synbiotrails.org
Predicting the sequence of an Inverse PCR product
This is a demonstration of a method for predicting the sequence of Polymerase Chain Reaction products. This approach is good for Inverse PCR, or other scenarios where the polymerase must "cross the origin" of the plasmid.
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Відео
Finding orthologs of a gene using BLAST searches
Переглядів 16 тис.4 роки тому
If you know the sequence of a gene and want to find other similar genes from different organisms, you can use BLAST searches. In this video, I demonstrate how to find the genomic DNA sequence for the ortholog of the dxs gene in another organism.
Visit Synthetic Biology Trails
Переглядів 4199 років тому
For more lectures and exercises on synthetic biology, visit synbiotrails.org. This online learning site will introduce you to the concepts needed to understand genetic engineering, and the current state of the art.
Construction Files
Переглядів 71510 років тому
A Construction File describes the lab operations for fabricating a genetic construct.
Formal Descriptions of Chemical Structure
Переглядів 37110 років тому
In this section, we will build up data structures to describe the state of a biological system. At any given time, you can think of a cell has being a complex data structure ultimately composed of atoms. There are changes in state of these atoms in terms of their position constantly occurring that is the mechanistic underpinning of the dynamic behavior observed in biological systems.
Object Oriented Programming
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In object oriented programming, "Objects" usually refer to bundles of information. For example, you can describe a Car as having 'number_of_wheels = 4' and 'license_plate = 4BIUO11', and this bolus of electronic information represents the physical entity in a computer. In these lectures, we will use Objects to describe the chemistry, design, and simulation concepts associated with genetic engin...
Essentials Concepts of Organic Chemistry
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Organic Chemistry is the domain of the physical sciences associated with the structure of carbon-containing molecules and their reactions. At the beginning of the field in the 19th century, organic chemistry was distinguished from other disciplines not based on the chemistry of carbon but rather on the study of matter derived from living systems. Ultimately, it became clear that the chemistry o...
Issuing commands in Clotho
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The Clotho command bar allows you to express instructions and pull up data.
Oligos received from IDT
Переглядів 1,4 тис.10 років тому
We've received the oligos we designed from IDT, so we can begin the experiment by running PCRs.
Three grades of water
Переглядів 35210 років тому
There are three grades of water: tap, house deionized, and doubly-deionized (ddH20). You want the highest grade, ddH20, for any reactions involving nucleic acids and for resuspending your oligos.
Setting up a PCR reaction
Переглядів 66010 років тому
Here we set up the PCR reaction in a PCR tube. We add ddH2O, phusion buffer, dNTPs, two oligos, template, and phusion polymerase. We then thoroughly mix the reaction by vortexing or slamming the reaction on the bench.
Spin down oligos before adding water
Переглядів 36910 років тому
Because the oligo material may become dislodged during shipment, it is wise to give the IDT-supplied tubes a quick spin before you open the lid.
Make 10uM oligo dilutions
Переглядів 1,6 тис.10 років тому
The working concentration for our PCR protocol involves 10uM oligos. From our stocks we have 100uM. So, we need to prepare 10x dilutions in ddH2O.
Putting materials away after PCR
Переглядів 13210 років тому
Put your enzymes, dNTPs, buffers, templates, and oligos back in the freezer when you are done working with them.
Running a PCR on a Thermocycler
Переглядів 1,9 тис.10 років тому
Now we run the PCR reaction by placing it in the thermocycler and running the 2K55 program. This is a timed program containing 30 cycles of denaturing, annealing, and extension temperatures. The particular program you choose will depend on the polymerase and size of the expected PCR product.
Pre-plan you tip usage to avoid errors
Переглядів 12310 років тому
Pre-plan you tip usage to avoid errors
00:02 Gillespie algorithm simulates individual reaction events using random numbers 01:03 Simulation of Gillespie Algorithm for stochastic data 02:05 Gillespie Algorithm simulates random events based on reaction rates. 03:07 Understanding elemental reactions and distinct species in Michalis-Menten kinetics 04:11 Updating counts of species when reactions occur. 05:10 Elemental rate equations for simulation 06:13 Updating species values based on reactions and probabilities. 07:20 Gillespie Algorithm records species counts over time. Crafted by Merlin AI.
The high frequencies on this mic are overwhelming and hurt my ears
Thank you! This is so much clearer and shorter than my lecture. I finally understood it.
Great video!
Good explanation of the basic principle
I know this is very well explained but I feel so stupid because I am not understanding a lot of what is said here.
What do you mean by "these modules do not necessarily correlate to polypeptides"? And what are polypeptide boundaries?
I hate my life. Not related to the video but something i wanted to share. I have no idea what's going on with my life and why i have failed the mandatory exam for three times already
it is not about how many times you fall but how many times you get back up. Do not give up on yourself yet
Thank you sooooooo muchhh!!! Your videos are awesome and helping me to wrap my head around molecular approaches! :) Thanks a ton and a half
These people are sick and should be put in a dark, cold prison for the rest of their lives. Including all scientists involved. Dont play with god or mother nature
fuckheads
My grandfather Al Vellucci was ahead of his time! 😇
How do we add the plasmid sequence ??? Antibiotic cassette
Your videos are so topic specific... they deserve 1000x likes for every view. Thank you for doing this.
please send me the references I want to learn more about pks
INB4 this video gets deleted "early days" "PCR" This stuff was banned in the 80's so they just changed the name. I guess we should skip the CRISPR enhancements and just jump straight into the face cream that makes your skin cells produce more "freckle" proteins
I would honestly says that his makes it seem more complicated than it is.
Wtf why are you in so much hurry
I’ve struggled learning how to until now, thank you so much!!
Thank you very much
Material transport system is secretion system too ?
I'm not sure what you are referring to
how can I calculate the time to the next reaction event? 6:46 you said 4 msec, but I don't know how you calculated it
The time of the next reaction is also randomly generated. It is adjusted to match a biophysically correct distribution of reasonable times, but it begins with generation of a random number.
This is a great video, incredibly useful.
Shaky camera on a top of your head is not the best way to go about creating useful content
It is well done, but it only occurs during bacterial replication which is important to highlight
Thank You
Thanks!
Thank you so much Sir
great video. Thank you
Thanks! This was very helpful and on point!
Amazingly done!
parce póngale voluntad. . . esas diapositivas son muy aburridas y la voz también es aburrida.
Thank you, this was helpful.
This video helped me understand a journal article I'm reading right now, many thanks!
Me too, unnatural amino acid encoding in e.choli. There have been amazing breakthroughs in the last few years.
I'm trying to model pathways between cellulose and furan polymers but had no idea what databases were available. It's especially hard to find information on molecule color (spectra). This was a great starting point. Thank you.
Why do we always represent DNA in the 5 prime to 3 prime direction?
It is just a convention. It has no intrinsic chemical meaning, it is just what people do. Polymerases write DNA in the 5' to 3' direction, so perhaps that was the motivation. But I don't know the history of this practice.
Can you go discuss the difference between type II and type V secretion?
Can you answer my question?
So nicely explained
Helpful video indeed. But eager to know the maximum length which can be deleted by this lamda red technique. If I want to delete a larger segment of the genome (more than 10 kbp) , will this work?
Yes, that should be doable, but may be more challenging. I would recommend constructing 500bp homology arms rather than just the 40bp oligo arms.
It's "Shea-Ackers" which is a model using systems of differential equations and binding polynomials to describe gene expression
do not just read your handwritings. try to explain. if you tell us just by some stuffs in this speed , we cannot still get what you say
@2:27: using shia-kers? Sorry, I didn’t get it.
1:05 are you sure is transesterification? looks like amide bond formation. 1:30 are you sure is transesterification? looks like thioesterase activity Thank you for the video
Fair point, I'm using the word transesterification a little loosely. The amino acid starts as the bare acid and is then phosphorylated to an adenylate, which is not strictly speaking an ester in the sense that the carboxyl group is attached to phosphate (PO-) rather than an alcohol (CO-). Often chemists will still call that an ester of sorts. When the amino acid is then transferred to a thiol on the protein, the product is correctly called a thioester, and the term transesterfication is typically used to describe the formation of thioesters as well as regular esters. The next step where the amino acid is transferred to the amino end of the NRP would more accurately be called a transamidation, but it is not uncommon for chemists to call this reaction transesterification. During cleavage of the NRP from the enzyme, different things can happen depending on what type of termination domain is on the NRPS. Some encode thioesterase activity, meaning the attacking group is water and the free acid results. Others do transesterification in the proper sense. Others do transamidation. And there are a few other mechanisms.
"a constitutive promoter is one whose observed states only include free and sigma-factor-bound species". Any reference you recommend fro more information?
There won't be any references making that statement--the genetics definition of 'constitutive' (from google) is "Refers to genes that are transcribed in an ongoing manner, with control limited to that directly associated with the metabolic state of the organism. Constitutively expressed genes, that is, are always "on"". Operationally, in bacteria this typically implies that only a sigma factor is involved, but it is not a hard-and-fast rule.
What are the best packages or softwares to run this kind of simulation?
COPASI is one that we use in courses at UC Berkeley copasi.org/ and there are others. The algorithm itself is not a lot of code and many folks just write it out.
You are awesomeee!!! please continue with those videos!!!
do the primers always come in nm moles when they are in lyophilised form??
I do not know if all oligo suppliers express the amount of material in the tube as nanomoles (nm). IDT does. If you were to synthesize oligos yourself, you would not have this number. Instead, you would measure the UV absorbance of a solution of the oligo, and calculate the molarity of the solution from this measurement.
@@lilpiggie2167 okay
How did you get quick multiplication by 10 again?
How much microlitre of 10uM have u prepared here?
That was 90 uL water + 10 uL of the 100 uM oligo stock
@@lilpiggie2167 ok so u prepared 100ul of a 10 ul reaction right?
@@ArnavPadhi It's 100 uL of a 10 uM solution
thank you! very helpful!
thanks!!!
A bit too quick, and I would have liked more information and explanation for someone not too well versed in biochemistry such as myself. But I appreciate your video nonetheless. Thanks