theCrux
theCrux
  • Видео 78
  • Просмотров 567 223
INs and OUTs of a CRISPR experiment (CRISPR KI vs CRISPR KO)
References/Resources: www.patreon.com/the_Crux
All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf
How to design a CRISPR experiment? How to perform surveyor assay to check gRNA targeting efficiency? How to perform a CRISPR knock-out experiment? How to perform a CRISPR knock-in experiment? What is the difference between the steps to perform a crispr deletion and crispr insertion experiment? What are the considerations for donor DNA design for CRISPR Knock-IN experiment? How to screen CRISPR edited cells? How to obtain clonal edited cell lines from a CRISPR experiment? This video is a technical and conceptual discussion around the practical design of a CRISPR...
Просмотров: 289

Видео

How to design and express CRISPR guide RNA [pX330 plasmid, Tutorial]
Просмотров 49514 дней назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf What is a typical CRISPR Cas9 protocol? What is the workflow of a typical CRISPR protocol? How to design CRISPR Cas9 guide RNA for a CRISPR experiment and clone crispr guides in a plasmid (pX330)? In this video, I discuss a typical CRISPR protocol workflow and prese...
Genome Editing with CRISPR-Cas9
Просмотров 2,8 тыс.Месяц назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf What is CRISPR-Cas9 technology? How does CRISPR-Cas9 genome editing work? What is the mechanism of genome engineering with CRISPR-Cas9? What is a PAM sequence? Where is the PAM sequence location and what is its function? What is the protospacer adjacent motif? How d...
CRISPR-Cas9-mediated Adaptive Immunity Mechanism in Bacteria [3 Steps of CRISPR-Cas system]
Просмотров 750Месяц назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf What is CRISPR-Cas9? How does CRISPR-Cas9 work in its natural context? How does CRISPR-Cas9 protect bacteria against pathogens by providing adaptive immunity? What are the different types of CRISPR systems? CRISPR-Cas9 is a Type II-A CRISPR system. In general, this ...
A Brief History of Genome Editing
Просмотров 449Месяц назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf This video briefly discusses the history of genome editing and how historical events have led to the current state of the art. The video discusses parallel timelines that have interwoven to result in modern methods today. It all of course rests on the discovery of g...
Protein Expression Vectors - tet-OFF and tet-ON (tTA/rtTA + Tet based gene regulation) [Part 5]
Просмотров 607Месяц назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf What are the types of tet expression systems? What is a tet-ON and tet-OFF expression systems? How do tetracycline inducible expression systems work? This video discusses the regulation of protein expression using a tetracycline inducible or repressible based system...
Protein Expression Vectors (pET vector) - Induction of Protein Expression (IPTG + T7 Pol) [Part 4]
Просмотров 9042 месяца назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf What is a pET vector? What is a pET expression system? How do pET vector expression systems work? What are inducible expression systems and how do pET vector systems work? What is the function of T7 RNA polymerase in pET vectors and how does T7 RNA polymerase contro...
Protein Expression Vectors - Examples (Fusion Proteins, Insulin, and Cas9) [Part 3]
Просмотров 4302 месяца назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf In this video, we explore some common protein expression vector designs and how they are constructed. These are helpful examples and applications of protein expression vectors to intuitively understand the protein expression vector construction. We discuss 5 example...
Protein Expression Vectors - Template, Codon Bias, Affinity Tags and Epitopes [Part 2]
Просмотров 3982 месяца назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf In this video, we explore how to obtain templates for protein expression vectors depending on the gene structure (intron and exon language). Prokaryotic genes are intron-less, whereas eukaryotic genes often have introns. Obtaining template for protein expression vec...
Protein Expression Vectors - Expression Host and Recombinant Proteins [Part 1]
Просмотров 6212 месяца назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf Protein Expression vectors are used to make protein from the template DNA. In this video, we discuss some of the common applications of protein expression vectors and the complications involved with protein expression when changing the host i.e. heterologous protein...
RNA Expression Vectors - Design, Applications, and Examples (In vitro Transcription)
Просмотров 3722 месяца назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf RNA Expression vectors (different from protein expression vectors) are used for the sole purpose of making RNA from a template DNA. In this video, we discuss some of the common applications of RNA expression vectors and their rules/principles of design. Finally, we ...
Golden Gate Cloning or assembly - Type IIS (BsaI) restriction enzyme based cloning
Просмотров 2,1 тыс.5 месяцев назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf Golden Gate Cloning or Golden gate assembly is a restriction enzyme based cloning that uses type IIS restriction enzymes. It is a powerful way to clone large numbers of inserts in a single reaction without the need of using multiple enzymes. This video discusses the...
Gateway Cloning: Simple and Multisite Gateway Cloning - LR and BP Cloning
Просмотров 2,2 тыс.7 месяцев назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf Gateway Cloning is a popular recombination based cloning method. It does not rely on restriction enzymes but instead uses integrases to recombine att sites between a final and donor vector. This causes an exchange of DNA segments that inserts the desired DNA into th...
TOPO Cloning - TOPO-Blunt, TOPO-TA, TOPO-directional
Просмотров 4,2 тыс.10 месяцев назад
References/Resources: www.patreon.com/the_Crux All videos on Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf TOPO Cloning (Topoisomerase-based cloning) is a commonly used method to clone PCR amplicons. TOPO Cloning bypasses the need of restriction enzymes and ligases. In this video, we discuss the three main types of TOPO-cloning systems - TOPO Blunt, TOPO TA, and TOPO dir...
Cloning with Restriction Enzymes (5 Levels) - Traditional Molecular Cloning
Просмотров 1,4 тыс.11 месяцев назад
References/Resources: www.patreon.com/the_Crux Playlist - Genetic Engineering: ruclips.net/p/PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf Molecular Cloning with restriction enzymes can be very easy or very challenging. This video discusses the fundamental considerations of traditional restriction enzyme based molecular cloning. We discuss sticky and blunt ends, the process of blunting, and how all of thi...
Plant cloning vectors - Disarmed Ti plasmid, Cointegrate vectors, Binary vectors - T DNA transfer
Просмотров 4,8 тыс.11 месяцев назад
Plant cloning vectors - Disarmed Ti plasmid, Cointegrate vectors, Binary vectors - T DNA transfer
Yeast cloning vectors - YIp, YEp, YRp, YCp, YACs - Shuttle plasmid vectors
Просмотров 5 тыс.11 месяцев назад
Yeast cloning vectors - YIp, YEp, YRp, YCp, YACs - Shuttle plasmid vectors
P1 Phage and PAC cloning vector - Pac sites, Cre-loxP, and SacB lethality
Просмотров 2 тыс.Год назад
P1 Phage and PAC cloning vector - Pac sites, Cre-loxP, and SacB lethality
F plasmid, BACs, Fosmid cloning vector - Fosmids are small BACs (Bacterial Artificial Chromosomes)
Просмотров 1,6 тыс.Год назад
F plasmid, BACs, Fosmid cloning vector - Fosmids are small BACs (Bacterial Artificial Chromosomes)
Lambda Phage vector, Cosmid cloning vector - spi phenotype and in vitro packaged lambda virus
Просмотров 7 тыс.Год назад
Lambda Phage vector, Cosmid cloning vector - spi phenotype and in vitro packaged lambda virus
Plasmid and Phagemid cloning vector - Selectable vs. Screening marker
Просмотров 4 тыс.Год назад
Plasmid and Phagemid cloning vector - Selectable vs. Screening marker
Dam and Dcm methylation (restriction independent methyl-transferases) affects restriction enzymes
Просмотров 1,8 тыс.Год назад
Dam and Dcm methylation (restriction independent methyl-transferases) affects restriction enzymes
Restriction Enzymes (Endonucleases) in Molecular Cloning
Просмотров 1,8 тыс.Год назад
Restriction Enzymes (Endonucleases) in Molecular Cloning
Genetic Engineering - Classic vs Modern methods
Просмотров 3,3 тыс.Год назад
Genetic Engineering - Classic vs Modern methods
RNA Polymerase III Transcription - Promoters, TFs, Initiation, Elongation, and Termination steps
Просмотров 4,1 тыс.Год назад
RNA Polymerase III Transcription - Promoters, TFs, Initiation, Elongation, and Termination steps
Prokaryotic Transcription Factors in Elongation and Termination (GreA, GreB, Mfd, NusA)
Просмотров 2,4 тыс.Год назад
Prokaryotic Transcription Factors in Elongation and Termination (GreA, GreB, Mfd, NusA)
Rolling Circle DNA Replication and Amplification - Plasmids and Bacteriophages (M13 + PhiX174)
Просмотров 11 тыс.Год назад
Rolling Circle DNA Replication and Amplification - Plasmids and Bacteriophages (M13 PhiX174)
Mitochondrial DNA (mtDNA) Replication - D loop Replication (deep-dive video)
Просмотров 13 тыс.Год назад
Mitochondrial DNA (mtDNA) Replication - D loop Replication (deep-dive video)
Telomere DNA Replication - Chromosome end replication problem in eukaryotes
Просмотров 6 тыс.Год назад
Telomere DNA Replication - Chromosome end replication problem in eukaryotes
DNA Replication in Space and Time
Просмотров 612Год назад
DNA Replication in Space and Time

Комментарии

  • @diyabhatt9127
    @diyabhatt9127 25 минут назад

    Thankyou for the explanation

  • @Wobbleb
    @Wobbleb 2 дня назад

    Respect 🫡

  • @SuryaprakashTripathy
    @SuryaprakashTripathy 3 дня назад

    Very nice explanation!

  • @georgeseese
    @georgeseese 3 дня назад

    Where is there an explanation for how this gene has a unique identification?

  • @earth2k66
    @earth2k66 6 дней назад

    Your voice is heavenly, and your videos are educational ASMR. Every complex detail is organised to perfection. I would rather watch your playlists for hours than read a book or articles for 15mins 🙏

  • @earth2k66
    @earth2k66 8 дней назад

    0:06 "Bacteria face routine harassment from Bacteriophages" 😂👌

  • @maxMax-q2c
    @maxMax-q2c 9 дней назад

    Thank you for the great videos.

  • @marlentoktomamatov5868
    @marlentoktomamatov5868 11 дней назад

    bro this is gold

  • @marlentoktomamatov5868
    @marlentoktomamatov5868 11 дней назад

    базара нет, шикарный видос

  • @EdT.-xt6yv
    @EdT.-xt6yv 11 дней назад

    Accelerationism/transhumanism/anthrophocene

  • @Middlebenchers-p4e
    @Middlebenchers-p4e 12 дней назад

    Sir please make a video on lytic and lysogenic induction in bacteriophage

  • @markambrozyak79
    @markambrozyak79 12 дней назад

    😏😏😏

  • @kianjazz
    @kianjazz 12 дней назад

    YAWA WALA NAKO KASABOT

  • @theCrux
    @theCrux 12 дней назад

    CRISPR-Cas9 in Genome editing: ruclips.net/video/7ESZTE6rjLI/видео.html gRNA Design using CRISPOR and CHOPCHOP: ruclips.net/video/r4puDgc9rBs/видео.html

  • @fabian_7264
    @fabian_7264 12 дней назад

    Is it beacuse the glucose from lactose is far from the membrane where adenyla cyclase is so it can enter glicolisis before reaching the membrane therefore the inhibicion is low, in the other hand when glucose enters thru membrane proteins it is very close to adenilate cyclase

  • @christianecampos5457
    @christianecampos5457 12 дней назад

    Very well made and extremely detailed video! It helped me understand the topic better so I could explain it in a test. Thank you very much, greetings from Brazil!

  • @user-rn3zu4no8q
    @user-rn3zu4no8q 14 дней назад

    Absolutely love your lectures, i have been reading genes by lewins along with the videos and i have to sometimes yours are even more detail oriented than them.

  • @biopharmmarkets2115
    @biopharmmarkets2115 16 дней назад

    I'm getting into yeast genetic engineering for a project I'm working on during my Ph.D. This is an extremely useful video on a topic that is not well discussed outside of review articles

  • @theCrux
    @theCrux 17 дней назад

    CRISPR-Cas9 in Genome Editing : ruclips.net/video/7ESZTE6rjLI/видео.html CRISPR Experiment Design and Workflow: ruclips.net/video/f0Q6JKu8Xjk/видео.html

  • @HarmeetSingh-jo3sg
    @HarmeetSingh-jo3sg 18 дней назад

    Superb vid!

  • @shanu6633
    @shanu6633 20 дней назад

    Thank you dear sir. Sir can u provide notes of your every lecture please that is very helpful for me.🙏

  • @somakhaled5487
    @somakhaled5487 20 дней назад

    thank you im student biotechcnology from jordan

  • @muniraabbaslimkhedawala3068
    @muniraabbaslimkhedawala3068 20 дней назад

    Can you please do cre lox p next please

  • @theCrux
    @theCrux 22 дня назад

    Erratum: A few errors have been pointed out since the upload of this video. 5:19 u and d should be swapped. BREd is closer to the TSS i.e. downstream of the TATA box. 8:55 TFIID is the biggest GTF (technically). TFIIH is a close second.

  • @elijahjohn4482
    @elijahjohn4482 22 дня назад

    @theCrux is the downstream and upstream part of the B reader element mixed up in your drawing? I am confused, I thought the BREd element is closer to the transctiption start

    • @theCrux
      @theCrux 22 дня назад

      Yep, they did get mixed up. BREd is closer to the TSS. This BRE u/d issue has been pointed out by others as well. *Time to add an erratum for this video*

  • @김대빈-w5m
    @김대빈-w5m 22 дня назад

    10:05 the attL site and attR site... the word L and R do not come from Left and Right, right? If they are, how does it suddenly come with two attL sites in one Vector and two attR sites in another? at 05:38 they were mixed as L R and R L after B B' , P P' recombination.. So I think R-DNAi-L and L-ccdB-R recombination will result back in P-ccdB-P and B-DNAi-B right? And also after LR recombination 12:26 the names attB1, attB3, attB2 are just randomly given? So it is also fine to name them attP1, attP3 and attP2?

    • @theCrux
      @theCrux 22 дня назад

      At 3:54 the L/R do come from Left and Right. At 5:38 the custom vectors contain B B and P P. At 9:57 the custom vectors contain L L and R R. At 12:26 the names are not truly random. There are attP and attB pairs called attP1/attB1, attP2/attB2 etc. Theirs products are likewise called attL1/attR1, attL2/attR2, etc.

    • @김대빈-w5m
      @김대빈-w5m 22 дня назад

      @@theCrux Thank you for you kind explanation! Sorry I think I confused you... What I meant was 3:11 you marked a half block as B and B' and also P and P'. At 4:52 you wrote B B and P P but just as hole block each. So my questions is the name B B and P P as block just mad up by yourself? Another question is, at 5:38 you wrote L and R in the donor vector (L-ccdB-R) just as you described at 4:01 (attL-virus DNA-attR). They have some combination (B+P'=L and P+B'=R) given that the whole block of B and P are divided into B and B' / P and P'. So at the end there are L-ccd-R part in the donor vector and R-DNAi-L part in the final vector at 5:38. But how come you write two vectors with (L L and R R) at 10:09? Becuase after B P reaction the Donors include either R+L or L+R (05:40). OR it is just made up by yourself to exlain LR reaction so it results in the final product as B B and P P? So it has nothing to do with previous stories of lytic phase or the initial BP reaction because after BP reaction the vectors have either L+R or R+L... I hope you understand my point. Thank you for your answer in advance!

  • @Middlebenchers-p4e
    @Middlebenchers-p4e 23 дня назад

    Thank you so much sir

  • @STn-vb5fj
    @STn-vb5fj 24 дня назад

    Really nice! I kinda get lost in all the names lol, I'd personally find these videos even more helpful if they went through the basic outline of the process in more depth, and then added names to what is doing what🙂i guess it wouldnt be so short of a video but still, great

    • @theCrux
      @theCrux 24 дня назад

      Thank you for this suggestion. I have experimented with the outline idea in some videos but those videos tend not to be as well received as jumping into details right from the start. Perhaps, I will come up with a way to make things better. Again, I appreciate you pointing it out :)

  • @АртемийКуришев
    @АртемийКуришев 24 дня назад

    Thank you for your talented presentation of relevant material

  • @tamarareed7581
    @tamarareed7581 25 дней назад

    Hi there! Quick question -- my professor said that DNA melting does not involve ATP hydrolysis but DNA unwinding does. When the AAA+ domains are involved, does that mean that ATP is being hydrolyzed? Or not always? Thanks! I really appreciate the time and effort you put into these videos -- they are fantastic.

    • @theCrux
      @theCrux 25 дней назад

      Hello! Both DNA melting and DNA unwinding are energy consuming processes. You are right AAA+ domains depend on ATP. For DNA unwinding, DNA helicase (which is a motor protein) requires ATP as well.

  • @Middlebenchers-p4e
    @Middlebenchers-p4e 26 дней назад

    Thank you sir

  • @edithaosman2216
    @edithaosman2216 27 дней назад

    You might wanna recheck the binding action of initiation factors. IF1 binds to the A site and IF2 with GTP interacts with IF1, the small subunit and initiator trna.

    • @theCrux
      @theCrux 27 дней назад

      Wow! Amazing error catch. I have updated the erratum comment on this video. Thank you!

  • @hurmatarif8342
    @hurmatarif8342 Месяц назад

    Thank you so much 😊 please make videos on DNA binding domain

  • @mattharvey8712
    @mattharvey8712 Месяц назад

    Bravo.........do one on insulin made from yeast.....cheers

  • @deepikakumar1430
    @deepikakumar1430 Месяц назад

    can you please elaborate on how primase and helicase physically separate

    • @theCrux
      @theCrux Месяц назад

      For E. coli, the interaction between primase and helicase is quite transient (at the order of seconds). The P16 domain seems to be responsible for mediating the transient interaction. For some other bacteria, the primase:helicase complex is stable for >30 minutes. Perhaps P16 (and the surface charge distribution for both primase and helicase) is the answer to your question, or may be something else. Unfortunately, I am not super up-to-date with the literature on this matter so that is the best answer I can provide at the moment.

  • @deepikakumar1430
    @deepikakumar1430 Месяц назад

    sir at 13.36 it is not clear, what do you mean by "DNA is not allowed to pass"

    • @theCrux
      @theCrux Месяц назад

      The DnaG allows the ssDNA to pass through it, meaning it scans the ssDNA for CTG. But as soon as the CTG is found and makes contact with the primase, that ssDNA is stuck and scanning stops (i.e. ssDNA is not allowed to pass through anymore).

  • @brokenpiano2251
    @brokenpiano2251 Месяц назад

    Thank you! I was looking for details on the orientation of sgRNA/target DNA, your video makes it so simple and clear. Also, I solved the exercise correctly :)

  • @Middlebenchers-p4e
    @Middlebenchers-p4e Месяц назад

    Thankyou sir

  • @mobagamer69
    @mobagamer69 Месяц назад

    Thank you for the video

  • @theCrux
    @theCrux Месяц назад

    CRISPR-Cas9 in Bacterial Adaptive Immunity: ruclips.net/video/I0aWfgvpCyU/видео.html gRNA Design using CHOPCHOP and CRISPOR: ruclips.net/video/r4puDgc9rBs/видео.html Design of a CRISPR Experiment: ruclips.net/video/f0Q6JKu8Xjk/видео.html

  • @asmrcalmcooking
    @asmrcalmcooking Месяц назад

    17:26 Why can it not replicate?

    • @geneie6050
      @geneie6050 День назад

      Because its ori is mutated to RNA pol can't bind properly to it

  • @Oopsnicolee
    @Oopsnicolee Месяц назад

    THANK YOU!!!!!

  • @АртемийКуришев
    @АртемийКуришев Месяц назад

    You are amazing!

  • @vaidhata
    @vaidhata Месяц назад

    Wow! This is exactly what I was looking for. Your ability to simplify complex topics while still retaining the details is truly impressive.

  • @theCrux
    @theCrux Месяц назад

    CRISPR-Cas9 Genome Editing: ruclips.net/video/7ESZTE6rjLI/видео.html Erratum: 9:20 RuvC domain blocks the HNH domain by blocking its active site. The stabilization of Cas9 in crRNA:tracrRNA complex induces the Cas9 to become competent. 12:00 R-loop refers to the RNA:DNA hybrid that forms when spacer RNA portion of crRNA tries to pair with the target DNA.

  • @Middlebenchers-p4e
    @Middlebenchers-p4e Месяц назад

    What a lecture sir

  • @Middlebenchers-p4e
    @Middlebenchers-p4e Месяц назад

    Kya video hai yaar

  • @theCrux
    @theCrux Месяц назад

    Erratum: 5:25 The loop is called Trombone loop, NOT priming loop. Some textbooks refer to them interchangeably, which is not correct. The priming loop is the loop formed by the moving replisome when it establishes a primer. For priming loop, see ruclips.net/video/a60aVk-Zumo/видео.htmlsi=tumKJKysR0V9wy8a

  • @unknownblackhole8789
    @unknownblackhole8789 Месяц назад

    I thought the priming loop was the loop formed by DnaG? It's the same as the Trombone loop?

    • @theCrux
      @theCrux Месяц назад

      You are Correct! Priming loop and Trombone loop are different. However, some textbooks refer to them interchangeably which is not true. The loop discussed here is Trombone loop, not Priming loop. Thanks for catching this. I will post an erratum on this :)

  • @Catherinezhang-v7m
    @Catherinezhang-v7m Месяц назад

    amazing!