Genetic Engineering

Genetic Engineering

  • Qu Tailong added an answer:
    Will the PCR of my DNA be affected if I do not follow the correct steps for DNA rehydration, as outlined by my kit instructions?

    I did a DNA extraction, but I am not sure if the rehydration step was good or not.

    The kit instructions told me to keep the DNA at 60°C for 90 minutes, but I only kept it there for 30 minutes.

    Qu Tailong · Hunan Agricultural University

    In my opinion,  you can get your DNA but the amount may be lower than in 90min.Most of the factors can effect the yields of your DNA,such as how many nts of your DNA, the amount of DNA in your original material.i have no idea what kit you used,actually it is a long time cost you 90 min getting your DNA .  you can check your DNA yields by PCR.if you have aplyfied, checkit by AGE.

  • Chunxin Wang added an answer:
    Are there general protocols that can be applied in most cases on how to carry out the optimization of sgRNA for CRISPR/cas9 method?

    It is known that the CRISPR method can have off-target effects if this is not well addressed by optimizing the sgRNA, taking into account that CRISPR has been used in genetic engineering from relatively recently, are there currently general "rules" for the sgRNA editing?

    Chunxin Wang · National Institutes of Health

    There is one paper reported that using certain chemical to help loosen the chromatin structures may enhance TALEN targeting. Some also argued that methylation may affect targeting efficiency as well for TALEN. I'm no aware of any similar reports with CRISPR. But since CRISPR is generally very effective, you really don't need to worry too much. Construction of CRISPR is so easy that you can just try several different gRNAs targeting different regions to find the efficient one. 

  • Sarwan Kumar Dubey added an answer:
    Can some one tell the rice varieties specially bred for Aerobic conditions or one can say Aerobic rice varieties?

    Most of the time varieties bred for wet lands are normally being used for aerobic and/or DSR conditions. I am interested in varieties specially developed to grow in Aerobic condition in semi arid part of India.Thanks.

    Sarwan Kumar Dubey · Central Soil and Water Conservation Research and Training Institute

    Hi Arshad Yaseen; Thanks for such an information. It may help me if I could arrange some seeds from IRRI seed Bank. Thanks

  • Mohsen Basiri added an answer:
    Why would one get a low fold induction upon doxycycline treatment?

    Has anyone used the FUW-tetO system for doxycycline-induced transgene expression? I transduced my cells with trangenes (MOI=5 each) under the FUW-tetO, then treated with 1 ug/mL doxycycline for 5 days. The fold induction was only like 1.5X to 5 X which seems very low when checked by real time PCR

    Is it normal for this system to give such low fold induction? or has anyone got a much better fold induction using this system?

    Thanks

    Mohsen Basiri · Tarbiat Modares University

    I agree with Jose that FCS tetracycline may be the problem and you can use Tet-free FCS or KOSR (if applicable for your cells).

    As Yoav said you need to coexpress a rtTA along with your transgene under FUW vector. This may lead to some considerations:

    • The induction rate (depends on cell type and virus titer) of both rtTA and transgene lentiviruses should be high enough to support duble-transgenesis of the cells. You also may use double antibiotic selection to isolate double-infected cells.
    • rtTA expression may be low due to cell type dependent weak promoter activity (I had such an experience with low CMV-derived rtTA expression in embryonic stem cells).
    • There is also a possibility of epigenetic inactivation of viral transgenes in certain cell types. This may be more evident at higher passage numbers and I believe to be the hardest obstacle to deal with!

    However, I don't want to freak you out, because these problems are not so evident and TET-ON systems are usually easy to setup and use.

  • Elma Mrehic added an answer:
    In DNA extraction from saliva, why doesn't the pallet dissolve in the tube after adding DDH2O?

    I am extracting DNA from Saliva through Manual Protocol NOT with the Standardized Kits. Right at the end of the extraction when I get the DNA Pallet that need to be dissolved by adding de-ionized water which normally does dissolves and we process it to further step gel electrophoresis. But I am facing difficulty in this step that is the pallet which does not dissolved. I am using Phenol, Chloroform, EDTA, Tris, BME and PK and Lysis buffer. Some of my colleagues ask me to wash the Phenol with TAE Buffer solution this will help you in proper extraction. Kindly help me out in this regard.

    Elma Mrehic · International BURCH University

    1X Tris EDTA buffer and leave the samples at 4 ° C overnight

  • Seeds DNA extraction?
    Which protocol is the best for extracting seeds DNA contents? With considering seeds of the poppy plant being used.
    Ketankumar Joitaram Panchal · Sardar Patel University

    You can try kits as well as conventional protocol but keep it in mind that what plants species you are targeting because poyphenol content will vary with plants species

  • Noha Said added an answer:
    What is the difference between PCR-tetra primer ARMS, PCR-AS, and PCR-CTPP?

    I want to know the difference between PCR-tetra primer ARMS, PCR-AS, and PCR-CTPP. I want to use the most simple one in the genotyping.

    Noha Said · Zagazig University

    THANK YOU

  • Rolf Henrik Nilsson added an answer:
    Is there any software or online tool to check the purity of gene sequence?
    How can we check the purity of the gene sequence (in percent)? Is there any software or online tool or any calculation?
    Rolf Henrik Nilsson · University of Gothenburg

    Some ideas on how to examine sequences for quality and reliability are presented here:

    https://www.researchgate.net/publication/235931750_Five_simple_guidelines_for_establishing_basic_authenticity_and_reliability_of_newly_generated_fungal_ITS_sequences

    https://www.researchgate.net/publication/235601668_Incorporating_molecular_data_in_fungal_systematics_a_guide_for_aspiringresearchers

  • Bhagya C T added an answer:
    Can any one please send me original papers on Genetically modified products.Thank you.

    I need original papers on Genetically modified products(successful or failed) example:Crops, Food products ,medicinal products.

    Thank you.

    Bhagya C T · Institute of Bioinformatics and Applied Biotechnology

    Thank you ...... Yuan-Yeu Yau.

  • Aldwin Anterola added an answer:
    Does anybody have whole sequence information of pART7 vector?
    I need whole sequence information of pART7 vector.
    Aldwin Anterola · Southern Illinois University Carbondale

    I found the sequence here:

    http://www.csiro.au/Organisation-Structure/Divisions/Plant-Industry/RNAi/Plant-vectors.aspx

  • Pradeep Kumar Brahman added an answer:
    What should be the DNA absorbance ratio so that it can be used in Genetic Engineering? (during purification of DNA)

    DNA absorbance ratio

    Pradeep Kumar Brahman · Koneru Lakshmaiah University

    Actually question is nor clear. In what kind of work that ratio is needed. If you run the UV spectrum of ds-DNA it gives absorption peak at 260nm. However if you clarify your applications I can help. Because I am also working on DNA analysis.

  • Chantal Agbemabiese added an answer:
    Why do my sequences have low signals at the 3 prime ends?
    I do a pre sequencing purification using EXOSAP-IT and I sequence using the Big Dye terminator cycle sequencing kit v.3.1. I then clean up using Sephadex G50 on a multi-screen filter plate before sequencing.
    Chantal Agbemabiese · Nagasaki University

    Thank you Pia. I was able to optimize my reactions finally.

  • Yuan-Yeu Yau added an answer:
    Can splicing occur when a bacterial gene is transfected in an eukaryotic system?

    Will splicing occur in a countinuous gene expressed in a eukaryotic system, if so, what steps can one take to prevent such potential problems?

    Yuan-Yeu Yau · Northeastern State University

    Most likely, if the bacterial gene sequence contains consensus sequences for an intron to be recognized by the splicing machinery in the eukaryotic organism.

    GUS gene is from bacteria. Researchers had inserted a plant intron into the gus gene and transferred into plants, and found out that the intron was efficiently spliced and gave rise to the GUS enzymatic activity (see attached paper).

    We had tried to study the expressing ability of several site-specific recombinase (SSR) genes from prokaryotes in higher plants. We usually scan those intron consensus sequences in the genes to predict the possible outcome. As John suggested, one can mutate out those intron motifs with site directed mutagenesis to avoid unwanted splicing.

  • Lingaraj Sahoo added an answer:
    How do trypsin inhibitors or amylase inhibitors introgressed in GM crops for insect resistance not affect us?

    Protease and amylase inhibitors are introduced into insect resistant crops produced by genetic engineering. Wouldn't these enzymes affect our digestive enzymes too??

    Lingaraj Sahoo · Indian Institute of Technology Guwahati

    This appears to be less of a problem in the case of human consumption as such crops would be cooked before human consumption, with concomitant protein denaturation and inactivation. However, such crops may also be used as animal feed so that potential differences between uncooked normal and transgenic crops should be evaluated. A study has addressed this issue by feeding rats with transgenic peas expressing high levels of α-AI1 and monitoring possible effects on intestinal metabolism, growth and starch and protein digestibility. The minimal nutritional differences seen up to a dietary level of 300 g per kg of transgenic pea should encourage the use of transgenic crops as animal feed.

  • Aarón Barraza added an answer:
    Do you know a gen or genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in Aspergillus niger?

    Focused to recombinant protein expression.

    Aarón Barraza · Center for Research and Advanced Studies of the National Polytechnic Institute

    I suggest to design primers to amplify the 16S rRNA to be used as a reference gene in Aspergillus niger for qPCR expression analysis.

    Regards

  • Lan T M Dao added an answer:
    Which are the mouse transcripts that are longer than 6kb?

    I'm finding a mouse gene which produces a transcript longer than 6kb to be the control for my experiment. Thank you for giving a name of it

    Lan T M Dao · Aix-Marseille Université

    Thank you all. I will try it

  • Xiang-Bin Wang added an answer:
    Has anyone used Trimer Codon Oligos and can offer advice?
    We want to create a semi-random expression library (around 30aa). This could of course be done by using poly-N oligonucleotides, but there is a newer technology where the oligo is synthesized using trimeric nucleotides (codons). This has advantages since you then can choose not to introduce stop-codons, avoid certain amino acids and make customized mixes of codons for certain positions in the oligo.

    My question is has anyone experience with these? How long oligo did you order? Any company to recommend?

    Thank you.
    Xiang-Bin Wang · HIAT: Hongye Innovative antibody technology Co., Ltd., Beijing, China

    we have ever constructed 7 libraries using the trimer codon. if yo want to order the trimer codon oligo, please contact me by email: CTO@innovabtech.com. I will provided you the name of the company.

  • Marcel Zamocky added an answer:
    How can I clone a gene and pet vector for heterologous expression?

    I am trying to clone gene of my interest along with tag to pET24 d (+) vector. As the insert has its own ATG, at the start and another ATG  in other frame which is showing restriction site for No 1, so if i design a restriction primer to isolate CDS from whole sequence it will ultimately truncate the CDS, so please guide me what strategy should i use to complete isolation and expression of CDS.

    Marcel Zamocky · University of Natural Resources and Life Science Vienna

    I can only confirm from my colleagues that Gibson assemby will work in this case. This is the easiest way, otherwise you need to mutate back the truncated part of CDS which is not always a straightforward method.

  • Nektaria Andronikou added an answer:
    How can I make efficient transgene expression in mice brain?

    Usually when we transfect the gene into this organism we sometimes get good expression but also sometimes bad expression. Is there any precaution or protocol to make most efficient transfection and gene expression?

    Nektaria Andronikou · Life Technologies

    Hello Pradip,

    Have you tried using an in vivo specific delivery reagent, such as Invivofectamine 2.0? Here is a paper where researchers did siRNA delivery directly to the prefrontal cortex.  http://www.jneurosci.org/content/32/13/4562.full

    I'm checking with the scientist in our lab who developed the product with regards to DNA delivery, but I would think this would offer a higher efficiency of integration.

    I'll reach out to you directly to discuss further.  Hope this is helpful.

  • Yuan-Yeu Yau added an answer:
    What book for basic microbiology in human? and what book genetic engineering for basic?

    What book for basic microbiology in human? and what book genetic engineering for basic? I wan to learn the basic but may be other can have suggestion..thank

    Yuan-Yeu Yau · Northeastern State University

    Hi Riki,

    Another one we use in school: Introduction to Biotechnology (3rd Edition), by Thieman and Palladino.

    This info is from Amazon (see attached book cover):

    http://www.amazon.com/Introduction-Biotechnology-Edition-William-Thieman/dp/0321766113

    "Thoroughly updated for currency and with exciting new practical examples throughout, this popular text provides the tools, practice, and basic knowledge for success in the biotech workforce. With its balanced coverage of basic cell and molecular biology, fundamental techniques, historical accounts, new advances and hands-on applications, the Third Edition emphasizes the future of biotechnology and your role in that future. Two new features—Forecasting the Future, and Making a Difference—along with several returning hallmark features support the new focus." 

  • Paulo Sérgio Monzani added an answer:
    Is it possible to select transgenic bovine embryos using antibiotics?
    I have done a kill curve using blasticidin and the embryos die (stopping the cleavages). I have put the antibiotic at the third day of development and I have analyzed at 7th day. When I inject the lentivirus particle that carries the blasticidin resistance into perivitelline space, the embryos survive the treatment with antibiotic, and by PCR, they are transgenic (apparently). However, if the embryos are transferred to the recipient, the calves are not transgenic. I believe that the PCR result is a false positive (Could the lentiviral particle carry the plasmidial vector and not the RNA?). Why are the embryos are resistant to blasticidin? I believe that the resistance gene could be translated without integration. Is it right?
    Paulo Sérgio Monzani · University of Northern Parana

    Dear Pavel

    First, I would like apologize for the late answer for you. I have injected the lentivirus particle at zygote stage with one or two cells, using a micromanipulator without a microinjection controller. Therefore, I do not have the precise quantity injected into perivitelline space. Alternatively, I have digested the pellucid zone at zygote stage and co-cultured the embryos with the lentivirus particle. In this stage, the embryos were in the first day of development. I wait one day for the DNA integration, and then I added blasticidin at the third day of development. Tthe blasticidin was added in the control (without lentivirus injection or co-culture embryos too). The control embryos died within seven day, while the embryos in the presence of lentivirus survived until blastocyst stage. Differently from the cells cultivation, in the presence of antibiotic, the cultivation of the embryos is impossible during ten days. I hope have elucidated your doubts.

  • Yuan-Yeu Yau added an answer:
    What parameters should be in the primer design when designing primer for xyloser reductase and xylitol dehydrogenase gene?

    I am designing primer for xylose reductase and xylitol dehydrogenase gene. Can anyone suggest me what parameters should be their in primer designing.
    If anybody having protocol please provide me. 

    Yuan-Yeu Yau · Northeastern State University

    @ Mahboubeh,

    Thanks for replying and mentioned the 'GC clamp' concept.

    Frank

  • Róbert Szabó added an answer:
    Can someone help with my RT-PCR to reduce unspecific amplification?

    There is more than ONE band during 2-steps reverse transcription PCR with a gene specific primer in my gel (Lanes 3 and 4) at higher molecular weights. I used revers primer in synthesize of cDNA. Except ncrease the annealing temperature, Can anyone suggest a solution to amplify only my desired gene in RT-PCR??? what about decreasing of cycle repeats and final extention?

    As you see, there is 3-4 bands in Lanes 3 and 4, but ONE band in lane 2. The template used in lane 2 is DNA and template in Lane 3 and 4 is cDNA. To synthesis first strand of cDNA, gene specific primer was used in Lane 3 and Oligo dT in lane 4.

    In fact the quality of extracted RNA was NOT good. Is it one of the reasons of observing more than ONE band??

    Many Thanks.

    Róbert Szabó · Slovak Academy of Sciences

    As many people wrote, you can check the secondary structure of the primers to avoid the hairpins or primer-dimers. You also wrote, it is a 2-steps RT-PCR. But I´m not sure if you mean nested-PCR. For the higehr specificity it is good to use minimally two pairs of primers and set 40 cycles in one and 25 cycles in second round of PCR. Otherwise you can check if you don´t have too many ambigiuties or inosines in your primer sequence. Too many  ambiguitues dilutes your primer and decreases a specificity, but I think that too diluted primer is not your situation however signal is good. It would be good to see the product of first PCR reactions and product of the second, in the case it is the nested PCR, of course. And the last thing which is good to avoid is to many A-T pairs at the end, where the synthesis of the strand begins. Good luck.

  • Akhil Raj P added an answer:
    Can anyone help with storage after heat inactivation before gel extraction?

    After heat inactivation of the restriction enzymes, is it possible to store the vector/DNA in -20oC for the following gel electrophoresis and gel extraction step? Or should I continue it immediately after heat inactivation?

    Akhil Raj P · Rajiv Gandhi Centre for Biotechnology

    Yes you can store, if purpose is only for gel analysis... if you are planning for an elution from the same gel, do not store plasmid for too much time (days)..

  • Francesco Santoro added an answer:
    Can anybody suggest an easy technique to screen transconjugants?

    After getting transconjugants how we easily screen them.. 

    Francesco Santoro · Università degli Studi di Siena

    You should first verify the phenotype by plating transconjugants on antibiotic containing plates (see attached publication), then you should confirm the presence of the transferred genetic element by PCR (and sequencing, if appropriate).

  • Daniel Lerda added an answer:
    How would you define "genetic modification"?

    Obviously a subject with a great deal of baggage attached to it, but try to forget that and focus purely from a scientific (genetic) point of view what defining features does it have?

    My interest in this comes from the recent debate here in the UK about whether mitochondrial replacement therapy constitutes genetic modification. Opinion is divided but the Department of Health thinks it is not. They say that GM is "the germ-line modification of nuclear DNA (in the chromosomes)" - specifically excluding the mitochondrial genome.

    See page 15 of this PDF file:

    https://t.co/FqItiLtYxs

    My own opinion is that this definition is not correct but, really, I am most interested to hear how other people (particularly the scientists on ResearchGate!) might define it.

    Many thanks

    Ted

    Daniel Lerda · Catholic University of Córdoba

    I agree with Eugene Daev it should be called "genetic correction"

  • Ravi Kant Upadhyay added an answer:
    How do you synthesize an alkaloid using an expression vector (plasmid)?

    I wish to know how to extract an alkaloid or any secondary metabolite by using Genetic engineering techniques.

    Ravi Kant Upadhyay · Deen Dayal Upadhyaya Gorakhpur University

    One can simply insert steroid responsive gene in vector, it can be expressed both in insect cells lines as well as vertebrate cell lines. One can induce programming by incorporating alkaloid pathway enzymes in plasmids. These inserts can be positively selected in colony hybridization assay, then apply blots to find its location, culture cells which will secrete alkaloids in the medium. 

  • Thomas Sternsdorf added an answer:
    Expression of prokaryote genes in eukaryotes.
    I'm having problems in expressing prokaryote genes in eukaryote cells. I am currently using Invitrogen's Freestyle Max CHO Expression System (anyone familiar with this system?). Transfected (lipofectamine) the suspension CHO cells with 37.5ug of pcDNA3.1 HisB plasmid with my gene of interests. So far there is no expression even though the cells grow (antibiotically selected).

    I am suspecting on promoter acetylation (currently working on it). I tried searching papers on prokaryote genes expressed in eukaryote system but only found only one publication which uses COS-7 and 293T cells to express MHC I molecules of Listeria monocytogenes (more paper suggestions are very much welcomed). My question is:

    Is the expression of prokaryote gene, cell-specific? Would it be good for me to shift to different cells i.e, COS-7 or 293T.

    I understand there is codon bias as well as protein toxicity to take into account. I can safely rule out the latter because the cells are growing under antibiotic selections. Any inputs and tips are deeply appreciated.
    Thomas Sternsdorf · Forschungsinstitut Kinderkrebs-Zentrum-Hamburg

    For your solubility problem. Many people create fusion proteins with SUMO-1 (or -2) to increase solubility in the procaryontic system. THe SUMO-fusion seems to somehow greatly increase solubility it might help with folding. if this would work for you that may help getting the protein inton the soluble fraction. HOWEVER: one important thing. should this be an option for you, you will have to mutate the C-terminal GG as you are working in eucaryotic cells which express SUMO-protease to cleave the SUMO 1 precursor to create the -GG-COOH. Another thing to try is the N-End rule, the amino acid after the methionine determines the half-life of your protein (A. Varshavsky Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12142–12149, October 1996). possibly this is another tweak to try. Generally speaking, there are probably as many reasons for poor ectopic expression  as proteins. Codon-optimizing was an excellent  idea to start with. PEST-sequences in your protein of interest could be destabilizing too. (Find using: http://www.expasy.org/tools/) try  treatment (8-10h) with a proteasome inhibitor (MG132, careful, toxic) to find out, if stability is an issue. If so, the treated sample will show higher amount of your protein.

    Good luck. 

    Thomas :-)

  • Aubrey Levin added an answer:
    Can help with a search for promoters?

    I'm looking the promoters of differents genes and I want to know if there are any specific database for this.

    I would appreciate any suggestions.

    Aubrey Levin · University of Toronto

    If you have specific genes in mind, search on Sciencescape for the field chart for each gene and look at the top ranking papers. You'll find a plethora of promoters, including the ones in the highest impact research.

    Here are a couple examples

    P53: https://sciencescape.org/field/130951347

    BRCA1: https://sciencescape.org/field/131070743

    Hope that helps!

  • Goutam Kumar Tanti added an answer:
    Will an inter-species fusion antibody be as effective as a wild type?

    If we fuse conserved region of antibody from mouse to a variable region from rat will the fusion antibody work with similar efficiency? Or is there any other method to murinise the sequence of Antibody raised in rat against a human protein. Actually I want to express an antibody gene in mouse which was initially raised in rat.. Please friends help me. Thank you so much in advance.

    Goutam Kumar Tanti · Technische Universität München

    Dear Annemarie and Dear William,

    This is an awesome help. Actually, I am worried for both as asked by Annemarie....Basically, I am trying to develop an autoimmune disease model of mouse so I need to use the mouse for long term...And that is why I am planning to make the chimera of rat variable region with mouse conserved region (Fc) and I am worried whether my chimeric antibody will be functional or not... But I am really thankful to u, its a very good discussion...Best regards

About Genetic Engineering

Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.

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