Science method

Genetic Engineering - Science method

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.
Questions related to Genetic Engineering
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is any body working in cellulase protein engineering?
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We have been using Bt toxins for a long time, if a situation arises that the targeted organism becomes resistant to that toxin will it be possible to kill the targeted Bt resistant insect with the chemical insecticide that we use to kill the non resistant one. And if not what are the steps we can take? Definitely one of the options will be more toxic insecticides which will result in killing of a broader section of non targeted organisms which will effect in disturbing the equilibirium of the environment. Are there any other suggestions?
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Actually development of resistance to a Bt can be "easy" because the resistant is to a chemical -- the specific Bt protein toxin in that Bt -- and has occurred in several lepidopteran and coleopteran species. Right now, in US, major resistance of Diabrotica (a Chrysomelid beetle) to Bt-Maize has been identified within only a few years after introduction of that GMO maize, in addition to the resistance of several important Lepidoptera to other Bt toxins. But in each case the resistance is specific to the particular protein, and can be overcome by use of another protein. Any given Bt will have one or more genes for the specific proteins (73+ have now been identified although the specificity of most is not yet known see Crickmore, N., Zeigler, D.R., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J, Bravo, A. and Dean, D.H. "Bacillus thuringiensis toxin nomenclature" (2012) http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/ ), the genes being located on a plasmid not in the chromosomal DNA. Each protein family, with its unique molecular conformation, seems to have a different receptor in the insect gut epithelial membrane. Thus cross resistance while possible is rare. And in all cases so far resistance in an insect population to a Bt does not result in cross resistance to "regular" chemical insecticides (organophosphates, pyrethroids, chloronicotinyls, ryanodine receptor antagonists, etc. But one CAN stay within Bt use by switching to another Bt, one that has different proteins, for example going from a Bt with a Cry1Ab toxin to one with a Cry1E or Cry1F See http://www.glfc.forestry.ca/bacillus/ for a database of Cry protein specificity based on bioassays or on molecular sequence
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Is it necessary to use any kind of Tag ? what alternative methods could be used?
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pET series vectors already have 6X-His Tag. Most probable way is to digest pET vector with BamHI and HindIII. Assuming that ur gene length is ~100 bp. Amplify ur gene of interest with having BamHI and HindII sites. Ligate ur gene with the vector and express in E.coli BL21 DE3 host, induce with IPTG and check on SDS PAGE for expression. Use Ni-NTA resin which can bind with 6X-His position . Then u can purify ur protein of interest. No second method is required.
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The first transgene will be easily screened by using marker gene analysis, but in case of second, 3rd and 4th genes it may give false results (positive results), then what alternative methods could be used?
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Hi Vikas
See the papers attached below where people have used strategies for cloning multiple genes in a single vectore....I hope some of them may be useful for you...You may be able to get some of the vectors from these labs
ajay
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I am trying to find a cis element such as a/ttttgxxrttaagg, in this case, is there any program on-line that can search my target sequence and find out the matched cis element? Thank you.
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Hey,
you could use matinspector (http://www.genomatix.de/online_help/help_matinspector/matinspector_help.html) and search for the corresponding transcription factor.
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I am researching in construction of recombinant luminescent strain, but know nothing about how to choose a suitable host cell. Any advice?
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The recombinant luminescent strain is constructed by converting a plasmid with sensing element and luminescent gene into a host cell, which results in a specific responce to environment pollution.
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I want to co-express two different genes in the Pichia pastoris, so as to catalyse a biological conversion from substrate A to intermediate product B, and to the final product C using the two expressed enzymes. I don't know which kind of plasmid can be used, and also want to know the way constructing the recombinant plasmid.
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Have there been any identified side-effects of transgenesis? Do you agree ethically with this biotechnology especially in animals?
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I am not sure what you mean by side effects. THere have certainly been issues that came up in teh anmals, for examples the original growth hormone pigs and sheep had serious problems due to the over-expression of GH. However, if you mean unintended consequences for the consumer, there is no evidence on htose lines being considered for use in agriculture that there is any isuue either for the animasl or for the intended consumer.
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Point mutation of ssrA gene is very very rare phenomena, cause which is ribosome rescue factor/molecule act as both tRNA and mRNA during error in translation, i done lot of attempt but i cant, I clone ssrA gene into multicopy plasmid and mutagenesis on cloned ssrA gene, now i want to introduce mutant fragment of ssrA in multicopy plasmid into chromosomal region, i cant able to do this, anyone suggest for this?
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I didnt try to do lambda-red recombinase experiment, ya sure ssrA gene is not essential in E.coli cause many known and unknown factors are there and its doing same function of ssrA as ribosome rescuing and tagging, It is not possible for introducing mutants on the ssrA gene in chromosome cause is not a protein its tmRNA molecules.
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I want to establish a cell or cell line to secret BMP4 permanently and I haven't any practical experience about this. First I need to know which kind of cell is appropriate for this purpose?
And I think I need appropriate vectors and cDNA that lead the cell to secret BMP4.
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I have published papers with my maiden name, and married name, however I cannot upload these papers because of the last name does not match the current. Can I add other last names (alternate) to my profile so that I can include these publications?
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Are there protocols out there, that could help me out with that task? Expression should be in human cells under the control of a minimal promoter, assay for promoter activity would ideally be GFP.
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This is called "shotgun cloning" or "construction of shotgun libraries"--and you'll find many Google hits with those terms. This is, for instance, how the libraries of DNA fragments used to perform the initial sequencing of the human genome were prepared. As with most things, the method of choice depends on the intended purpose. Do you want very large segments containing full length genes or do you want small fragments which might have detectable promoter activity if cloned into an expression vector?
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I have to clone and express a viral gene in a mammalian cell line so I'm searching for a shuttle vector including eukaryotic elongation factor 1 alpha (eEF1alpha) promoter with multiple cloning sites plus neomycin & ampicillin resistance gene for selection. Does anyone have experience with such a vector?
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EF-1 alpha is also widely used for gene expression by transfection as well as CMV promoter.
I performed lots of innate immune gene expressions using EF-1 alpha promoter purchased from invivogen.
Here are literatures that describe EF-1 alpha promoter is more active than CMV promoter in some conditions.
Comparison between cytomegalovirus promoter and elongation factor-1 alpha promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein.
Promoter-dependent EGFP expression during embryonic stem cell propagation and differentiation.
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The mutagent for mutation was gamma rays...
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I have done plant the 3rd generation, but why still show large variation in their performance?
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My gene has been sequenced from genomic DNA of the target species and I already have tried altering Tm, Cycle, Duration of each steps, cDNA+Primer+Mg salt concentration. the reagents are totally ok.
I extracted the RNA with Trizol and RT kit was invitrozen. What could be the problem?
Thanks in advance.
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Hi Ahsan
Your reagents may be fine but what is the basis for you conclusions for your gene of interest...have you checked your cDNA using some control genes or reference genes like actin, GAPDH etc. or any other gene which you may have worked previously...
If other genes are amplifying it will indicate that the cDNA synthesis is fine ...but that still does not tell you why the gene of your interest is not amplifying....there can be several reasons for that..e.g, low expression levels of your gene of interest is one of them....so you need to rule out the problems one by one..
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I designed a construct of VP16 activation domain fused with a HD domain of a transcription factor (the transcription factor is on the C terminal fo the VP16) in an expression vector. However, the fusion protein did not seem to initiate transcription activation. Does that mean that VP16 activation domain is not enough, that additional domains of VP16 are required for recruiting other factors for transcription? I'm puzzled with how this system works.
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I find it difficult for citizens to get the best or the worst out of GMO's due to the unfavourable discussions I have come across. Help me out somebody, is it good or bad?
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Direct genetic modification of plants is simply another tool in our scientific toolboxes. I feel that if we use it responsibly and carefully regulate the testing and distribution of GMOs, they could be our best chance at feeding a world in which droughts and floods and rising temperature seem to be our new norm. That said, I agree that many of the business practices of companies producing GMOs are reprehensible, but I would argue that this has nothing to do with the GMO itself or the technique, and everything to do with profits and ethics (or lack thereof). We can't allow unscrupulous behavior by GMO producers to blind us to their intrinsic potential, or conversely be convinced that they are the cure to all of what ails our crops everywhere. By all means, we should pursue GMO production, but at the same time do so carefully and with an eye towards the greater good and the possible consequences of our actions years from now, rather than simple on next quarter's profits.
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I am looking for a protocol for DNA shuffling ?
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Hi, I'm trying to do a 5' RACE but I'm not getting a clear product. It is very smeary and the amplicon is not even the expected size. I am using the "5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0" from Invitrogen. Primers (GSP1 and GSP2) have been tested on genomic DNA and work properly. I suspect that my template cDNA concentration might be too low.
After SNAP purifying the reverse transcribed cDNA, I nanodropped my sample and it showed 5-8ng/ul, which seems very low. Perhaps the transcript I'm trying to amplify is not expressed at high levels? Does anyone know what the template cDNA concentration of a sample before PCR should be in a successful 5' RACE reaction?
Thanks
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A picture of your gel and details on how the smear differs from the "expected" size would be useful. And "expected" based on what--do you already know where the transcript(s) originate(s)? I assume that you are doing nested PCR from the cDNA. There really is not much you can tell from a nanodrop read--successful PCR of a specific product is the only meaningful endpoint. One possibility is that the gene is highly expressed (some independent assessment of this would be useful) and there are multiple diverse transcript initiation points. For some eukaryotic (again, I assume) promoters, transcription may initiate anywhere within a region of ~100bp. You can try diluting your cDNA before you start the nested PCR phase of your 5'-RACE protocol. At the proper dilution point, you would see only (zero), one or two bands corresponding to specific transcript start points. See protocol details in attached manuscript.
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I am planning to do a site directed mutagenesis in a 1.8 kB gene. I wish to mutate one amino acid at a time to study its role in enzyme catalysis. Can someone suggest good and affordable kits that are currently available?
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You dont need any kit for it. Just buy Pfu Turbo taq. it is a bit expensive, but you can buy according to your need. then you need DpNI that is not very costely, you can choose any company. Design primers by using Quickprime primer design tool at Stratagene webportal. Some times, you may modify your primers manually as well. You can get the PCR program and use of Pfu turbo. It is very simple and easy. If you want to see the details of method, see my thesis
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Have a look at the special Nature Collection on TAL Effector Nucleases:
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How to enhance production of antibiotic in medicinal mushroom?
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I need a fungal gene synthesized, but I'm having trouble in choosing between the existing companies. I've been Google searching, and found two companies in the US that I took in consideration: Biomatik and Genscript. Problem is that I haven't found any customer reviews about them... I was wondering if any of you have some past experience with these companies, or if you could suggest a different company which is good (and maybe cheaper as well) for this purpose. Much appreciated!
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Geneart experience: Price is OK $ 0.28 / bp, but Geneart tends to add some baloney charges for complexity at checkout (even though sequence is NOT high GC or AT, repetitive, or anything weird, no predicted strong secondary structure either). Then I got an email after ordering that my order would be delayed by 10 days because of the holidays (in MAY, for chrissake? wtf? They synthesize in Germany, Germans have X-mas in May?). For another order, a labmate of mine tried to order a codon optimized viral gene, but was informed that she would have to do mountains of paperwork plus it would take 2-3 months to complete. The hammer: they tried to sell this as a plus, because she would then have all this paperwork ready for other things. Yeah right. Had better experiences overall with Genscript. They are faster, no BS charges, and no other baloney.
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Hello there,
I am interested in a gene from sheep, CCT8 (chaperonin containing TCP1, subunit 8 (theta). However, I don't know if it was already sequenced, so I am trying to identify and annotate it manually. I blasted a protein sequence from bulls against ovine sequences. A lot of unknown cDNA sequences from ovine came up. Some of them showed 100% of identity. Which sequence should I choose? Which tools can I use instead of clustalW?
Thanks. Appreciate any help.
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Thanks a lot for your feedback David, I am gonna check it out at Ensembl. You gave me a lot of ideas.
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I have been working with whole-genome expression data using ESCs and embryonal carcinoma cells, but due to the size of the datasets, I sometimes struggle to easily identify particular markers of differentiation towards dermal layers and markers undifferentiated cells. Other than the typical gene annotation tools (DAVID etc), which I find often misses certain potential marker genes or produces false positives, I haven't found any other trustworthy repository of marker genes besides the tedious process of comparing them to previous papers.
Would anybody know a quicker and easier method of identifying marker genes in the data sets?
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Hi Sebastian,
I do not know which array technology you have used (Affymetrix, Illumina, Agilent...), but most of the time manufacturers provide software to compute their own data. Most of the time, you remain unsatisfied because these tools do not go as deep in analysis as you want to.
Happily, there are other tools :
- first, Partek : http://www.partek.com (today the web site seems to be down, retry later) To explore whole expression data. Powerful, easy to use and a free trial can be download.
- Gorilla : http://cbl-gorilla.cs.technion.ac.il/ to view GO dependances
- Genosplice : http://www.genosplice.com/services/rna-analysis an online tool where you can upload your data and do some analysis
- dChip : http://biosun1.harvard.edu/complab/dchip/ a software (download) with a lot of parameters, powerful but not so easy to use at the beginning
- BCGenEx Miner : http://bcgenex.centregauducheau.fr an online tool, from my research team, not on your cell skill (here it is breast cancer), but maybe can help you to explore cancer mechanisms.
This list is not exhaustive, but I hope it will help you.
Wilfried
Natural selection
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Does the evolution of beings by natural selection necessarily show that they are intelligent beings?
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Ok! Let me add one more bit in the discussion. The evolution of living beings, based on natural selection, is possible due to the quantum nature of the genetic combination, where the probability of having different combinations is not zero. Genetic variation gives rise to mutations, which depending on the environment where they happen, survave or not survive (natural selection). The mutation, although probabilistic, is correlated with the environment and depends strongly on the selection. For me, natural selection occurs in environments that do not undergo changes introduced by specialized intelligent agents. Intelligence is survival. Definition of intelligence is an intellectual matter.
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Update basic genetics conceptions-comprehensions. RNAs are Earthlife's primal ORGANISMS... Life Is Simpler Than They Tell Us http://universe-life.com/2011/08/31/origins-in-cells-clusters-intercell-cleanup/ Evolution: Genes to Genomes to Mono-cellular to Multicellular Organisms; Direct Sunlight to Metabolic Energy, Too; Tryptophan to Serotonin to Melatonin to Neural System. (cue for inter-cell cleanup time) A. Tryptophan to Serotonin to Melatonin Melatonin is a hormone secreted by the human pineal gland during night-time darkness. It is now marketed in the US as a nutritional supplement. The hormone is an indolamine compound derived from the essential amino acid L-tryptophan, with serotonin as an intermediate precursor. Tryptophan is one of eight essential amino acids not produced by the body but coming from the diet. The additional fourteen amino acids are produced metabolically. In the brain, tryptophan converts to serotonin, the neural-transmitter. Tryptophan is the only source for serotonin in the brain. Insufficient L-tryptophan in the diet is a cause of many severe biological malfunctions. Some serotonin is converted in the pineal gland to melatonin, the hormone involved in inter-cell processes during sleep time. B. Sunlight to Metabolic Energy Bio-clocks are products of the innate sun-dictated active-inactive pattern of genes and genomes, parents of Earth’s life. During life genesis and its early evolution direct sunlight was the only source of their usable energy. This situation persisted well into the evolution of the early mono-cellular organisms, and both genes and genomes display, therefore, innate “inactive-sleep” phenomena. The incorporation of mitochondria with some cells initiated the metabolic bio production of bio usable energy and furnished the evolving mono-cellular organisms with new, additional, flexibly available local energy. This development opened up a variety of courses of evolutions of cultures of mono-cells communities. C. Individual Mono-Cells to Cooperative Mono-cells Communities, Cultures As individual independent genes aggregated to cooperative genes communes, organisms, genomes, so individual mono-cells aggregated cooperatively into mono-cellular communities, cultures. http://universe-life.com/2006/03/22/natural-selection-at-cellular-level-life-is-a-cooperative-affairs/ “Life has always been and still is a fractal affair, repetition of phenomena on ever more complex scale. It cannot be otherwise; it evolves. And surviving-proliferating life has always been a cooperative affair since cooperation is most successful for overall survival/proliferation.” Cooperation requires all sorts of interactions, including maintenance, protection and foraging for food-energy. Organisms’ interactions are “cultures”. Cultures require “cultural energy”. Melatonin and some proteins are dark-and-light cue signals evolved by the mono-cells communities for timing inter-cells processes when the intra-cells processes are at “sleep-inactive” state. Melatonin is a derivative of serotonin a derivative of tryptophan, and proteins are genes’ tools, energy-dependent metabolism products. D. Mono-cellular to Multi-cellular Organisms: Mono-Cells Culture to Neural System, to Nerved Multi-Cellular Organisms Now we can appreciate the fractal nature of life’s evolution. It is ever-continuous ever-enhanced ever-complexing cooperation. Now we can understand why, and grosso modo how, all the organs and processes and signals found in multi-celled organisms have their origins in the mono-cells communities. And this includes the functions of serotonin and melatonin and, yes, the evolution of neural cells and the neural systems with their intricate outer-membrane shapes and functions and with their high energy consumption requirements. Now, circa four billion years after initial genesis-evolution with direct sun’s energy followed with evolution with also indirect, bio, sun’s energy, some of Earth life, we humans, find ourselves short of energy and in need of exploiting again more, and more directly, our sun’s energy… Dov Henis (comments from 22nd century) http://universe-life.com/ http://universe-life.com/2011/07/10/seed-of-human-chimp-genomes-diversity/
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I like to study about genetic distribution of plants.
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Genetic distribution of plant species can be determined through analysis of population genetic structure by using molecular markers such as RAPD, ISSR, SSR, DNA sequences, etc. After that PCR products were scored as present (1) or absent (0) binary characters and data matrix can be analysed by using GenAlex 6.2 software (Peakall & Smouse 2006), POPGENE Version 1:31 (Yeh et al.1999), or ARLEQUIN (Website for software and manual: http://lgb.unige.ch/arlequin/ ). Genetic diversity at population levels differentiated by number of effective alleles, percentage of polymorphic loci. Analysis of molecular variance (AMOVA) was used to partition the total variation into components within populations and among populations. We can also calculate gene flow, geographic location that showed genetic differences, quantification of variation and relationships among populations.
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Baculo virus transfection
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IVRI izatnagar, Bareilly
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maybe M. tuberculosis
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Antisense phenomena is generally absent in bacteria. But u can knock down gene using Sigma targetron kit and is effective for M. tb. Another way is using suicidal vector. Clone gene of interest in suicidal vector and introduce in native strain. If the protein corresponding to gene of interest is prest in bacterial, the bacteria will survive else it will die.
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maybe M. tuberculosis
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Antisense phenomena is generally absent in bacteria. But u can knock down gene using Sigma targetron kit and is effective for M. tb. Another way is using suicidal vector. Clone gene of interest in suicidal vector and introduce in native strain. If the protein corresponding to gene of interest is prest in bacterial, the bacteria will survive else it will die.
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Assuming that we have a good negative control, and the right melting temperature, what can cause very low fluorescence level of the melting curve?
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Are you running the reactions until the amplification reaches the plateau phase? You don't have to do that to get the Cq value, but generally more product will get you more signal. If you are reaching plateau and still have little fluorescence, it could be your sybr green is not fresh, as mentioned above. If you are limiting your primer concentration, you may simply run out of primer before you reach a very high fluorescence, this is also true of any other reagent like dNTPs. Of course, using a smaller reaction volume, you will have less of every reagent, and you will have less fluorescence, eg) I had less fluorescence when using 12 uL reactions than when I did 25 uL reactions.
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Which of the plasmid copy number detection protocol can help? Protocols on net ? Can anyone specify any ideal protocol? Please send some recent reviews or papers even in animal system if you have..A good statistical explanation is what I am also looking for..
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I think it is a good and reliable method for the detection of copy number if you have information about the copy number of the reference gene, One can carry out the analysis using the genomic DNA
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I'm looking for sources of Type III recombinant human collagen.
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Try at Europa Bioproducts, cat.nr. RPNKX-940.
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Does anyone know software or online tools that could help me make predictions of a protein sequence. I would like to test whether it would be a possible transcription factor.
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You can use this online tool, PANTHER (Protein Analysis Through Evolutionary Relationships) Classification System. It is freely available.
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I need a pBS-SK-Sfi cloning vector. It would be a great help if anyone can provide the same to us.
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pKD3 and PKD4
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Coli genetic stock center yale university
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Can someone help me with the oil <fatty acid> producing strain of alagal species? For example, Botryococcus braunii and its fatty acid producing strain and its properties.
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Please go through attachment
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I'm currently trying to figure out if anyone knows of any data on the transcription start site of the human U1 promoter in case of the missing initiator box. Does the -50 element still define the location of the start site in this case? Is the purine rule still valid then?
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Hello. I used to work on U1 transcription initiation during my thesis (which mean before 1993), and for as far as I remember removing of the -50 PSE element complitely abrogate transcription and moving the PSE element lead to change in the initiation start site. For experimental details you can have a look at Lescure A., Murgo S., Carbon P. and Krol A. (1992) "The proximal promoter and the start site cooperate to specify correct U1 snRNA transcription initiation by RNA polymerase II" Nucl. Acids Res., 20, 1573-1578. As you will dee in this article, initiation always take place on a purine.
Best regards, Alain
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What protocol do you follow to get the best results? Thanks in advance
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Protocol for the Blue White Colony Screening or Selection
Composition of LB Medium:
1.0% Tryptone
0.5% Yeast Extract
1.0% Sodium Chloride (NaCl)
pH 7.0
Batch Protocol
Dissolve 10 g Tryptone, 5 g yeast extract, and 10 g NaCl in 950 ml ultra pure water.
Adjust the pH of the solution to 7.0 with NaOH.
It may not be necessary to adjust pH every time, since pH of LB naturally close to pH 7.0. This might be affected by your reagents.
Add 15 g agar.
Complete the volume to 1 liter.
Autoclave on liquid cycle for 20 minutes at 15 psi.
Cool the media to 50 C in a water bath.
Add 1 ml of X-Gal.
Add 1 ml of 100 mM IPTG Solution.
Add antibiotic for selection.
Mix well and pour prepared LB agar into plates.
Dry opened LB plates at room temperature under UV light for about half an hour.
Individual Plate Protocol
Add 40 µl of the X-Gal Solution (20 mg/ml).
Add 10 µl of 100 mM IPTG Solution.
Spread evenly on the plate with a sterile spatula.
Dry the plate at 37°C.
Spread bacteria evenly on the plate.
Incubate overnight at 37°C.
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Mechanism involved
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The most common means is by homologous recombination. Typically this requires that the DNA which has been transformed into the organism has a region of homology to the chromosomal DNA.
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What would be the best technique for in vitro cell fate mapping, when the duration is about a month? What different protocols or products are available?
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Although I searched online, no information was found.
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I was trying to purify some ChIP DNA following several histone modification IPs.
After reverse crosslinking the DNA fragments, I added RNaseA to it, incubated for at least 30 minutes, added Proteinase K with Tris and EDTA and incubated at 45C for over 2 hours. This procedure/sequence of steps has worked before and it's the protocol I follow. Following this, I did a phenol:chloroform:Isoamyl alcohol extraction, followed by a chlorofom extraction. Then I proceeded to precipitate the DNA using glycogen (2µL at 5µg/µL) and 2.5 volume of 100% EtOH.
I saw goop precipitating in some of the tubes I was working with. I treated all the tubes exactly the same, so I cannot comprehend why some of the tubes formed this goo precipitate where as the rest did not. After centrifuging, I removed the EtOH supernatant from the tubes and let the pellet dissolve in 0.1X TE. I'm thinking about repeating the RNase A, Proteinase K treatment and the Phenol chlorofom extraction.
My question is, when repeating the process again, should I be adding more glycogen for the DNA to precipitate, more salt, etc.?! How else do you think I can recover my DNA fragments? Will repeating any of the process I listed above affect my yield?!
Thank you so much for your input! :)
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Remember to wash the pellet with 75% ethanol if you wish to add more salt. The salt may hinder the following steps.
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I want to transfer the GFP protein gene to my cell lines. I want know the simplest way to do so. Preferably a chemical method.
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Every time I was doing allele replacement experiments, I met this problem. It seems that one of my homologous arms didn't work during the two recombination process. Due to it, all I got finally were wild-type ones, none of mutated-type.
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The band may be of 100bp. Can anybody suggest the standard protocol for RE Digestion?
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You may try following reasons
1. Low initial DNA used for digestion. Increase initial DNA.
2. Fragments produced are low in concentration. concentration may be increased by ethanol ppt
3.Increase agarose concentration 2%, As 100 bp fragment travel very fast.
4. Run bromophenol blue dye indicator only half a distance of gel as it separates approximately around 500bp and your fragment will be seen much before it.
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I am looking for PCR conditions to amplify this gene.
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I never worked with that vector, but I have some experience with molecular biology, so let me try to help. Are you sure that the primers were designed properly? Make sure that the primers are specific for the flanking regions of the gene you want to amplificate. If you're sure of that, take into considaration that the times and temperatures of the cycles must me adjusted to the size and G-C content of the target gene. The higher the G-C content, the higher the denaturation temperature must be. The time of the elongation cycle depends on the size of the gene. If you need more information, please send me more details, and I can help you if you think it's necessary.
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Can anyone recommend a seminar topic?
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Good suggestions, additional comments to point out: 1) verify that the gene specific over expression is showing same physiological effect, or at least close to, that of the endogenous genein the cell. Ideally you want to do a titration (different plasmid conc. for example) of your over expression and chose the lowest conc. that gives desired physiological effect; 2) similarly you'd like to verify that the interaction between your overexpressed gene and its endogenous homolog doesn't introduce undesired effects. You can try cell lines that do not express endogenously the target gene if possible; 3) finally, because the ectopic gene expression doesn't follow the endogenous pathways, there's always a possibility that some protein specific modifications (important for gene function) may not occur.
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Dears I want to produce drug in plants (Molecular farming) by expression my gene in lettuce. i have a challenge, it is enzymatic degradation in the stomach. my question is " Are there any amino acid sequences that protect my drug in stomach?" can i attached this peptide to my protein? Best regard
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I think you are moving too fast here. if i were you, i will first engineer the drug in the plant, work out how well the drug is expressed, under what conditions etc. and once the dosages (optimal expression levels) are determined, then now you can start to worry about delivery. i.e. Whether the drug is absorbed at high or low PH, the position of absorption along the GIT. With this knowledge you can then determine whether to simply inhibit digestive enzymes before your drug is eaten to enhance uptake or to protect your drug from digestive enzymes among others..
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I'm studying the population of soil microorganisms present in the rhizosphere. I used DGGE to investigate whether differences exist between populations of healthy and decayed trees. I've used the Pearson's Correlation to build the similarity matrix and UPGMA to construct the dendrogram.
Now I would like to select some samples to sequence the V6 (for bacteria identification) and ITS II (for fungi identification) region but I do not know how to do it. Or better ... I have no way of justifying why I choose certain samples.
Does anyone have any idea how to select samples for sequencing based on data obtained from DGGE (Denaturing Gradient Gel Electrophoresis)?
Thank you
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I agree with previous answers that you should select multiple samples, and possibly all samples, and do a barcoded 454 pyrosequencing run. Your DGGE data might show that certain samples look different, but other samples might not be distinguishable using that (lower resolution) technique. So you could be confident to pick samples that are different, but it would not be certain if the samples that look identical on DGGE are really identical. If you only have a couple of hundred of sequences, I would advise combining them all in one pyro run. Using the Hamady/Knight lab approach, we have combined up to 300 different samples in one full 454 run. It will still give you 3000-5000 sequences per sample, which is pretty deep sequencing!
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hey guys. I am willing for a training in rDNA techniques in India. Please get me the updates, if any.
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Friends
I srihari, requested you to get latest updates about restriction endonucleases. If you have any body please send me to sriharigunji@gmail.com. please
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I have a question related to sequencing analysis. Does anyone can give me an idea for exp. if I found the same species in my sequences is it good or not to do the phylogenetic three ?
One more question is that how could we know about the revolution of gen by using sequencing analysis or another method is you know ?
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reasons may be different! you are right! MAybe sample is not pure! that the compound conсentration of sequence mixture is not suitable. Thus high DNA concentration inhibits primer correct job and sequence rection . Primer didn't bind properly. pay attention to the electrophoresis cleanliness electrophoresis, if you checked DNA after isolation and PCR. Proteins, RNA and other outsider agents can inhibit sequence rection too. Or the resolution for the peak is not proper due to background noise and ur data needs to be normalised. Re-purify and check again! good luck!))
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Hi,i have completed B.Tech in Bio Technology,during my final year project me and my friend found one complete new remedy for diabetes from INSECTS,by animal studies we confirmed.We got 1st prize in an International Project Competition and Exhibition held at Vel tech college on Feb 2011.We PATENT our project in Intellectual Property of India, our C.B.R. No-7180.We want to study MS in abroad,Could anyone help us?
If i find right professor i will be grateful to you people,If anyone knows a professor who is working on diabetes project or going to start diabetes project kindly inform me. We are ready to come any part of the world to study and research. Thanks you people who all are read my post.Take care.
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hello Pradeep
lots of congrats..plz would u send me a copy of ur research?
thank u so much
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Dear friends,
Im using cell designer for pathway drawing. I finished it. Then i have to analyse the path. For that i need to import the .xml file into cytoscape. I cant. Some error was occuring. anybody had any idea , please help me...
or suggest me some tools for metabolic network analysing...
Thank you.
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which is most effective method for seperation of protein and why?can all the protein being seperated by all the method.
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one quick way that we use is promiscuous affinity chromatography. for example, dextran sulfate cellulose beads are great for isolating proteins which express Lysine and/or Arginine residues on their surface. you can then do step/gradient elution with increasing ionic strength solutions. proteins with few basic amino acids exposed usually come out in the wash volume so we wait until the optical density is below ).005. Collect the wash and check for you protein. if it is in this volume. stop. get a lysine-affinity get from BioRad or Lysine Sepharose. or make a poly-lysine affinity gel. If your protein is still on the DSC column start your step elution. If you are isolated your protein from plasma always diluted 1:1 with H2O before you apply it to the DSC column. Your first step in elution should be to was with PBS, we like to include MgCl2 at 5mM. do the same if you have to resort to a Lysine Affinity gel column. dont forget to add protease inhibitors from the start. tell me about your protein and i will try to help. use jguevarajr@rgv.rr.com
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talk about biotechnolgy in Colombia is so difficult, because research isn´t as important as talk of poverty, inter conflict, drugs and narcotrafic corruption, because there are the principal troubles in my country, the research in this topic is less avanzed than other countries as Chile, Brazil and Mexico, our action field is limited, there isn´t companies looking like such a biotechnological profile but rather looking for people from other areas to apply biotechnology i am engineer biotechnology now, how is biotechnologies studies or research in Colombia?? this for wind our action field.
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Kelly, I can tell you our experience. In my country, Argentine, the biotechnology has many histories about their initiation, maintenance and develops. It depends on the subject, that is, soya development at the end of the ´90 (with all the OGMs development that it implies), pharmaceutical biotechnology with an strong enforcement in vaccines production. Finally, the first transgenic animal obtained in our “pampas”!
The development of soya and others OGMs were possible by the pressure of the agroindustrial sector. The other was the result of a political decision enhance by the international power, it is the BID and other credits from international banks to the research area.
it is important to participate in organizations like FAO (REDBIO Colombia) or others that it could support and make pressure to develop this subject in your country.
Patricia Marconi
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can all the target dna combine with all type vector or have specific vector for recombination
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the ligation of target DNA and vector depends upon the presence of restriction sites..... if vector ends have complimentary restriction site to the target DNA it will surely ligate...
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my question is that is it possible in the biotechnology to produce a virus with the desired properties .. for example if some one need some receptors or any other molecule or... Show moremy question is that is it possible in the biotechnology to produce a virus with the desired properties .. for example if some one need some receptors or any other molecule or channels or pores which are not normally present in the natural viruses .. answers from the experts will be appreciated...
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just answering according to best of my knowledge....
As viruses are link between living and non - living, so they themselves can't be engineered to get a fruitful product... In order to do so they need to be expressed in host cells and bacterial machinery proves to be efficient for that. In that case antigenic properties can be introduced in host so virus expresses some receptors against it which can be identified and isolated efficiently..
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i want to do my main project in PG.. so please tell me..
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hi... im from Malaysia. I really need help from u all. I cant find any journal about dna barcoading for aloe vera. now, i just refer to dna barcoading for land plant. actually.. i m am 3rd year student of chemical engineering in bioprocess and now i need to do final year project. my project is about development of dna barcoading technique in identifying aloe vera.
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Im very sory about late reply, hida_89@yahoo.com.
Tq
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This relates to drought stress in the tropics.is using Rec. DNA Tech therefore an acclimatization to the high temp stress?e tropics.is using Rec. DNA Tech therefore an acclimatization to the high temp stress?
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its a good concept but whatever the rec. strain you develop, it must be highly sustainable in S.African Climate.
Journal of Pakistan Medical Students
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How does the virus know that it does need to mutate or is it just random? Are there known internal factors within the living organism are the direct cause for mutations?
Why does a substitution in nucleotide base pairs take place?
Or perhaps non living mater that is near by as part of the life support medium of the living organism?
I’ve been asking myself for a long time and perhaps many others still are interested in the Virus mutation mechanisms, more like the factors that produce such event(s). Here, also as branching out the question, I propose to discuss about the biological transmutation of elements phenomenon.
Enzymes suffer transformations, modifications, mutate. Viruses do mutate.
There are mechanisms, the internal or some external clocks and / or some physical events with chemical reactions implications that take place I believe.
Are isotopic activities possible factors to trigger virus mutations? Is it the same for enzymes?
Perhaps we should look at it closer to be able to identify not only possible triggers but also to better understand the mechanisms that mutations take place in their complexity.
Or perhaps we have a combination of isotopic activity and other activities that interact?
If any isotopic activity is involved, what are the isotopes we may talk about or have under observation? What are the atoms we may look closer at?
What is known, proved or at least of hypothetical nature in this area of concern?
I hope I made myself understood as is not really my line of specialty.
Thanks to all members in advance and perhaps we can work something out, a research project may be borne out of this as team work? It is multidisciplinary complex subject that perhaps is worthwhile to try to unveil.
Adrian Toader-Williams
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Mutation is random in evolutionary theory 
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can any one explain the role of genetic engg
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Genetic engineering plays a major role in developing transgenic organisms with altered and improved characteristics by introducing a new gene or by silencing a particular gene... In the field agriculture, benefits from genetic engineering which has improved the genetic fitness of various plant species. The common benefits are increase in the efficiency of photosynthesis, increasing the resistance of the plant to salinity, drought and viruses and also reducing the plant’s need for a nitrogen fertilizer. It can help in improving the quality and quantity of particular characters in plants and animals and microbes...
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can any body tell me the size of this gene.
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it is say about 26 strands i meant the twisted ones :)
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You can win in open innovation challenges. If you have expertise and innovative ideas you can win open challenges posed by international companies and get $1000 to 5000 on an average. It is easy and free to register. Copy and paste the following link on your browser bar:
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Is there any "Lycopene" gene construct available in public domain ?
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not yet....
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Dear friends,
I need the of GFP and cellulose gene constructs, can any one help me in this regard.
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Hello, I'm wondering since blackhat hackers abound on the internet, and have plans for even RFID transmitters in national IDs, and break into everything electronic and non (like lock picking) and are experts at social engineering, how nanobiotechnology will deal with the first human/machine interfaces, especially in the area of the brain. I was going to be a whitehat hacker for a while (defense) but it wasn't my vocation and was mostly curious about certain aspects, but basically, a human interacting directly with the Internet or computers, even stand alone, stand the chance of becoming "infected" as well as chips inserted in not just the brain but other areas, when anything from video games to other software have come out with virii on the manufactured DVD/CD, directly from the company. We definitely need safeguards, as well as "firewalls" for our brains and even DNA since that is our code/blueprint for healing, growing and procreating and adapting basically, since our bodies change genetically each day per reading material I've read. Also, spies, terrorists etc could be passed easily down the line genetically if implants are somehow used to change genetics, creating tendencies and even memories. I remember that some dreams/memories or near death experiences that are common are the results of what is in our genes. Even info could be passed on this way similar to hiding code inside a photo, the human body/brain is technically a computer/machine. http://www.tricksystem.com/2008/12/secretly-hide-any-file-inside-jpg-image.html IF someone makes the connection and basically translates the machine code to human code, it's like converting from hexadecimal to base 10 in comparison. Also, stenography is basically how to hide messages and with distributed computing etc and botnets, perhaps people could be controlled or manipulated easier and is dangerous for all, especially military or scientists. Religion already shows that masses can be manipulated (especially when used improperly) as does media and entertainment such as music, movies and even books. The effects of music alone are hypnotic and activating someone, even involuntarily is not impossible nor extremely difficult. Think of something akin to the Manchurian Candidate but without direct involvement per se and no blame except against the individual. Best of luck, am tired and ill and going to rest but I have more ideas and activating individuals, since we're bio-electrical can take as little as radio waves, cell phone microwaves, EMF, an MRI etc. BTW, anyone interested in funding my education? I thought of this with a mere associates in Telecom and 3 semesters until my Telecom. I'll tell you that Ray Kurzweil is one of the people I admire and I've books of his that I checked out and also own "The Age of Spiritual Machines" since he's into nanotech as well as A.I.
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Dear friends, This is mohandev from Andhra university visakhapatnam, India and I need a cellulase gene for my work. How can I get that cellulase gene hardcopy and can anyone is provide that consrtruct to me ? please help me i this issue
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can anyone please tell me what is the sensitivity (% level) of detection of gm foods by PCR when they are contaminated with Non-gm foods ?
(primers cam 35s, nos, EPSPS )
please mention recent articles possible.
Thank you all !
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in fact i look for a linker for hybridization of two distinct gene. how can i find it?
Thanks.
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which protein your interested to hybridize is important. The Linker we use usually is a stretch of 25-30 AA which don't form a secondary structure i.e. beta sheet or alpha helix. It is usually rich in P and T which make it flexible to make interact the different domains proteins
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Isolation of Plasmid
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I highly recommend you using the book "Molecular Cloning: A Laboratory Manual", especially Volume 1 (newer editions are way better). They have many protocols with different reactives, and they also explain the use of every reactive. In general, it's an amazing book and you will find several variations that may be helpful when you don't have certain reactives.
Does anyone know the prevalence of the Mx gene in Egyptian native poultry breeds?
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Fayoumi
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Does it mean the shape, size and the intensity?
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Thanks a lot!
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Genetics
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There are different approaches to genetic transfer, but personally I would recomend you a viral vector, because virus have all the genetic machinery needed to make an efficient transfer. The type of virus depends on the type of cell that you are going to infect, my view is that a lentiviral vector is your best choice, due to its RNA genome. I recomend you to read a good review and the user´s manual of a lentiviral system (Clonetech of any other brand).
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Low weight molecular proteins are easy to be degraded in E. coli :(
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May I ask you that did you solve this problem already? 
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We know that the heterochromatin assembly in S.pombe is guided by RNAi, But if we are looking for a gene on euchromatic region i.e. on normal chromosomal locus what would happen?
A question is also raised if we silence a gene of primary metabolism then what would be the fate of cell longevity.
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if the gene is essential for the survival (primary metabolism will be essential, i guess), then it will be difficult to silence the gene because that will be lethal for the cell. but if the cell survive, it can give you insight into the possible roles played by that gene.
On the other hand, there are some methods of inducible repression of a gene, which you can try for ( controllable promoter or inducible knocking down).
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With some examples
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Hi Siddharth..
One can join a gene of interest with a blunt or sticky end vector..but aprt from the 'procedural technicalities' it is also decided by 'what is the objective after cloning'..and both the approaches have some 'pros and cons'...
1) Blunt end cloning does not require particular enzyme for the insert,,,you can use a proof reading polymerase for amplification,,,and if it is a restriction digested fragment...one can use an enzyme to make it blunt. (differnet approach for overhangs or recessed ends)...so you have less dependance on the compatibility of enzymes used for digestion of vector and insert....in this approach one can also use adaptors to make sticky end fragments 'blunt' ....However, ligation efficiency of blunt ended fragments to a vector is low, and not all the enzymes do blunt end ligation ... and you get less transformants...
2) If you have options to get vector and the insert digested by sticky end generating enzymes ...it should be best..the efficiency of ligation is good, more enzymes are available for sticky end ligation and you get more transformants....
3) some things are decided by your objective...you just want to clone ...or you want to clone and express.....so there is no dearth of possibilites in both the approaches for cloning a fragment in a vector....I hope you got some information
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Introduction for primer designing.
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Primer 3 is simple and widely used program, has many different input parameters that you control and that tell primer3 exactly what characteristics make good primers for your goals.
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I kept DNA samples (undiluted) which I isolated about 3-4 months back and preserved at -80'c and after 2 months I kept them at -20'c. Yesterday I checked them on gel. Almost all of them show shearing.. I did not save samples also. ..Any suggestions...Do they give amplification on PCR...
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Hi...How much sheared are your DNA samples...it may or may not work..depending upon what are you trying to amplify..Amplification of small fragments or multicopy fragments is still possible as the shearing is random...long fragments and single or low copy regions may be a little bit difficult....I suggest you give it a try with one or two samples first...If you can send a gel photo to have a look at the samples it would be nice
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This series of articles traces the development of Genetic Engineering from its biological roots through to its biggest applications. The journey starts off with the elucidation of the principles of heredity in the mid-late eighteen hundreds and ends at the cutting edge of genetic engineering today. A. R. Chakravarthy invites you to make that journey.
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@Chris Wilkins Thanks for sharing wonderful and informative articles on history of genetic engineering..
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Knowing the fate is to make preparedness or unwanted worries about likelyhood of onset of particular illness or diseaes.
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Any information (including genotype of an individual indicating future complications) if used in a 'RIGHT manner' is useful...if not, there can be so many problems...
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I have a grant to develop protocol and stably transform our local tomato varieties for disease resistance. I am in search of a suitable laboratory where such work can be done, preferably where bench fee is not charged. The grant covers flight ticket and subsistence.
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Dear Akinola
If you intrested to work with us, please feel free to contract me with all the details regrding your grant etc......
Who has the vectors, pCL112 and pCL113?
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These vector can be used to test protein interaction by BiFC.
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It could be Land snails DNA and gene extraction.
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Check online. But if you need tutorial follow me on twitter
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Dear all..
I've finished to culture and isolated the pET-15b-56ST-GFP Plasmid... now I'll try to cut the GFP gene from pET-15b-15b-56ST-GFP plasmid, but I don't know with what I supposed to cut it... because there are so many cutting site in there, so I still confuse what the better one of restriction enzym I supposed to used..??
I want to put the GFP gene to pBI121 plasmid as one way easier to predict the gene that we put to the plant is expressing in it... but the matter is I still dont know how to cut the GFP gene, what the restriction enzym that I supposed to used??? can anyone help me to answer it, please!!
Best regard....
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Eko send me restriction map of your GFP and pET plasmid. I have map for pBI121. I'll tell your which enzymes you can use to cut GFP and ligate it into pBI121. send your restriction map on this address sarfrazkiani786@hotmail.com
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if we try to inter GBMs emboli into arterial cerculation(animal models) could they cascade itravascular and adherte to brain vascular ednothetium and make changes in the microenvironment as systematic metastasis and open BBB to survive in the parenchyma causing metastasis
or auto-immunity suppress and kill cells ,although melanomas have the same genitic origin can cross our immunity system and make metastasis,Farther abscess have the keys to open BBB.
thanks.
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Gfp in flower
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This is my project title; i was wondering what specific things should i take into account?
So that a lot of info could be found
-thanks-
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are they useful? I am assuming they are a by product, no? could they be harvested for some other use? could reverse protomics be used to trace the origin of the misfolding? is there some structure or organelle that could house these misfires?
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The banana cv. I work with grows 14ft. in height
Can anyone has a gene for inducing dwarfism in banana, how to get it?
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Hi Srinath,
1. If the 'height' of banana is controlled by complex genes (several genes), it won't be easy just to introduce one gene to magic the work, and make them become short.
2. The semi-dwarfism gene (Sd1) from rice has been reported (see below), which conferred 'Green Revolution Rice'. You may want to work on this kind of gene and find out; especially nowadays the Genome-editing tools are so powerful that you can knockout a specific gene to check out the mutated phenotypes.
**This is the article: The genes of the Green Revolution [ http://www.sciencedirect.com/science/article/pii/S0168952502000094 ]
(From its Abstract) The spectacular increases in wheat and rice yields during the ‘Green Revolution’, were enabled by the introduction of dwarfing traits into the plants. Now, identification of the genes responsible for these traits shows that they interfere with the action or production of the gibberellin (GA) plant hormones. We knew that the wheat Rht genes encode growth repressors that are normally suppressed by GA, and recent work shows that the rice sd1 gene encodes a defective enzyme in the GA-biosynthetic pathway.
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When I run the crude plant extract in SDS-PAGE, I see a clear gel with no protein bands at all... kindly suggest me some ways.
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K thank u for u r suggesion. I will try it
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Since hyposia can increased red cell production. I think we can just check if suppression of red cell production under exercise can do this.
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If i would like to clone a marine metagenomic library and i would use E.coli as a host for the preparation of metagenomic library then in that case there is chance that i would miss the expression of particular gene function bz it may be possible that it will nt express in E.coli, so how to over cum ths problem????
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Hi everybody,,, any one knows which genes controlling boron absorption and translocation in plant?, specially brassicaceae family
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Are some cultivars of wheat with genome of the species T. macha?