how to make liposomes for gene dilevery?
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The lipid parts have a cation binding site to bind with the negative charges of the DNA.  The final complex is called a liposome.  \n\nIt then carries the DNA into cell and into the nucleus.  You still have to use a bunch of reagents in the process.  This is just from my research background.  The specifics are long.  You can email me for them
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Go here. http://www.dnacenter.com/\n\nYou can't make them get it done, but you can trick them into giving you DNA by collecting a straw they have used or a cigarette butt or, obviously, a semen sample. Anything with hair, skin cells, or spit will also work. There is no age or time limit.
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Go to answers.com and type molecular mutagensis at search you get links go through them.
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The main question is: do you want to make transient or stable transfection. If you want to do stable transfections, then you will need a selective marker such as resistance to G418. \n\nSome vectors also code for a fluorescent protein such as GFP or add a tail to your protein such as a His Tag. This helps for the visualization of your protein or the purification but beware of potentially inactivating additions.\n\nYou will always have a polycloning site so what you want to find is a set of enzymes that you have in the lab in a good amount and work with them. Find the vectors that have their restriction site for the insert.\n\nAs for the promoter of use, ask around in the lab for what works the best with your cell line. It's not such a big choice.
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http://www.reference.com/browse/crystal/23235\nhttp://www.reference.com/search?q=nucleic%20acids
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Introduction. This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as "knock-out" mice or "gene targeted" mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact Thom Saunders with any questions or Meet the Staff.  The Transgenic Core prioritizes all requests for service on a "first-come, first-serve" basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received. \n2. Plan the experiment. What is the purpose of your experiment? Do you want to completely ablate gene expression in all tissues? Do you want to analyze tissue specific regulatory sequences? Do you want to produce a model for tissue-specific gene deletions? You will need to obtain or clone the structural gene that you want to mutate. The source of the DNA is important since published studies demonstrate genomic DNA isogenic to the embryonic stem cell line give higher gene targeting efficiencies in ES cells (see Key Gene Targeting Papers). While complete sequence information is not necessary, a detailed restriction map of the gene is an irreplaceable tool. During the planning stages the material transfer agreements for R1 ES cells and other reagents should be initiated so that they are ready in time. Once a genomic clone of the gene is in hand and restriction mapped, then it is time to... \n\n3. Clone and the Verify the Targeting Vector. The Core has cloning vectors for investigators to use in gene targeting research. They include pPNT, a basic targeting vector, pNZTK2, designed to insert the lacZ gene behind the promoter of the targeted gene, and pflox, which is designed to introduce loxP sites into the gene. A complete materials transfer agreement is required before they can be distributed. You will need to determine which 5' and 3' fragments of the gene will be used to construct the targeting vector. Generally speaking, a couple of Kb on either end should give a reasonable gene targeting frequency. You will also need to consider the methods and probes that you will use to identify ES cell clones that have undergone homologous recombination with the targeting vector. This means a Southern blot approach with probes outside of the targeting vector or a PCR based assay with primers outside of the targeting vector. Once the orientation of the fragments in the targeting vector are confirmed and the probes are tested then it is time to... \n\n4. Electroporate ES Cells and Select Recombinants. ES cells are a very sensitive reagent, and most often the weakest link in the process of producing gene targeted mice. Fastidious tissue culture technique is necessary to maintain the totipotent state of the cells during electroporation, selection, and expansion. In order to maximize successful outcomes, the Transgenic Core maintains ES cells and companion reagents that are quality tested by producing ES cell-mouse chimeras that transmit the ES cell genome to offspring. Investigators may choose to use the Core's Gene Targeting Service (we do all of the tissue culture work, you do all of the molecular biology), or to use space in the Core lab (rent space in our completely equipped laboratory for ES cell culture work), or to do th
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Doesn't get much simpler than this website, have fun!\n\nhttp://www.biology4kids.com/files/cell_main.html
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Easiest method involves the pooling of the source (best source is wheat germ due to ease) next grind the germ then add some detergent solution- this will lyse the cell wall (the grinding breaks down the plant wall) next is to add some form of alcohol (isopropanol or high % ethanol is best (91% Isopropanol is available at drug stores)) Add the alcohol and the DNA will float to the top with the solution- spool on a glass rod.
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Yo man i shall give u a few sites that wil enable u to design the animal cell and then fill in the design with the items u believe will suit.\n\nhttp://www.batesville.k12.in.us/bhs/science/paper_bag_cell_model.htm\n\nhttp://www.batesville.k12.in.us/bhs/science/sponge_cell_model.htm\n\nhttp://www.batesville.k12.in.us/bhs/science/styrofoam_cell_model.htm\n\nhttp://www.cellsalive.com/cells/animcell.htm\n\n\n\n\n\nfurther details: mng_1432@2die4.com
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Sure.  You say that the cells are competent.\n\nJust clone your cDNAs into your plasmids, and then perform transformation (chemical or electrical, your choice).\n\nElectrical transformation's more fun, though I have caught a specimen on fire that way.\n\nBacteria aren't picky about which plasmids they intake, just make sure that the plasmid contains a bacterial replication promoter so that the bacteria knows to duplicate it while performing mitosis.\n\nAlso, plasmids in excess of 12kb length have trouble getting into bacteria, so be warned.\n\nHope this helps.
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order from Invitrogen or some biotech company for a bottle of DMEM...thats what most labs do nowdays.\n\nWhen preparing a DMEM for mammalian cells, you should have Foetal Calf Serum or Bovine serum and some antibiotics (e.g penicillin/streptomycin/glutamine) depending on ur cell type.
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First of all make sure your template is good quality. Did you extract genomic DNA, do you have a library in plasmids...(?).\nTry re-purifying your template.\n\nMaybe your template is GC-rich or has some secondary structure.\nIs your template very long?If so make sure you let the polymerase is working at the elongating temperature for enough time (1min/kb)\n\nIf you are using plasmid template try not to use too much. Don't put more than 30-40ng DNA in a reaction. \n\nCheck your primers:\n-can they form a dimer?\n-can they anneal at more than one site on the template DNA with a only few mismatches?\n-is the difference in the Tm of the two primers more than 6 C (it shouldn't;ideally they should be the same and I try not to exceed a difference of 4)?\n-do your primers end in more than 3  c or g in a row?\n-what's their Tm and GC content?Tm should not be very low (I design my primers at 56-61) and GC 40-60%.\n\nThe most strigent conditions are using as annealing temperature 2 C below the lowest Tm of the two primers. Try lower temperatures.\n\nYou can try different enzymes Taq, Pfu, Pwo, etc, even mixtures. Not all enzymes have the same processivity.\n\nYou can try different Mg+2 concentrations.\n\nThere are a lot of control experiments you can do depending on your template and primers. Eg if you have a library in a  plasmid that has sites for universal primers you can use one such primer and the other respective primer you designed in order to check if there is something wrong with the quality of your primers. \n\nIf everything is perfectly designed and you've tried evreything but still get a very faint (but of correct size) band, gel purify it and use it as a template for a new PCR. In this case you'll have a very high chance of getting mutations so you'll need to use a high fidelity enzyme like Pwo or Pfu turbo and of course sequence a lot till you get a correct clone.
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I jsut did the same project with my 13 year old son.\nThis is what we used\n\nhttp://www.enchantedlearning.com/subjects/plants/cell/anatomy.GIF\n\nwe had to make a model
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the dna in your cells has large bands of useless areas called introns (intervening codons) within large bands of useful areas called exons (expressed codons).  if you transcribe these to make RNA, the resultant product is called hnRNA (heterogeneous nuclear RNA).  hnRNA won't be useful because it has the useless introns imbedded within it.  to make it usefull, our cells splice out the introns leaving only the exons in the final mRNA.\n\nso if we want to create, say, a baceterial plasmid that can produce insulin or some other protein, we couldn't just take our genes and put it in the bacteria because bacteria don't splice out the introns.  the way to do it then is to transcribe our genome, splice the hnRNA, then with the resultant mRNA, reverse-transcribe it back to DNA.\n\nthis DNA that we made won't have the introns from before in it.  now this length of DNA can be used to make proteins without the need for splicing.\n\nThe next step is to put this DNA in a plasmid that can be used.  This plasmid may have all kinds of genes on it, such as resistances, binding sites, cutting sites, etc.\n\nWhen you combine all these different sequences together, the end product is called recombinant DNA because it came from more than one source.
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NaCl needs to be at the correct concentration, as it is needed to ensure that enzymes etc. work correctly.  It allows the cells to have the correct concentrations of sugars etc by influencing water entry by osmosis.\n\nA cell can take up a linear plasmid, but probably won't be able to propagate more copies if is isn't annealed, unless the correct DNA Ligase enzymes happen to be available in the cell.
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Try this , it really helped me in my first few lectures in cell bio , such as DNA and RNA . Loads of animations and stuff ! \n\nhttp://www.johnkyrk.com/DNAtranscription.html
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Let me bump yo butt like a machine of laundry, yeah.\nPumpin out my love like a mitochondria.\n\nMove it up and down just like I told yee,\nThen fold it up like a big ol' Golgi.\n\nShe'll double-bomb ya with a combo hip-titts,\nLike the bond of phospholipids.