Good hygiene is also required to guarantee the minimum chance of crosspollination amongst plants by insect vectors. If the plant material that is being grown is transgenic, then it is the responsibility of the researcher to hold any necessary licensing and to abide by the national legislation for the growth and disposal of these lines. Measures should be in place to allow for the removal of material, where experiments are put at risk by practise of others. This strategy works best if someone is appointed to be in charge of the area, such as a glasshouse manager, who can monitor the growth area and act appropriately.
Before starting to grow Arabidopsis, purge the glasshouse area or growth room to remove all potential infection. Infection can be spread between growth areas on clothing, so do not move between glasshouses and growth rooms in the same laboratory coat. Avoid leaving bags of compost open to prevent contamination with fly eggs and fungal spores. Either of these regimes can encourage infections. Arabidopsis can be grown on soil, commercial compost mixes, perlite, and other relatively inert media. However, there are certain criteria that have to be matched. Drainage can be enhanced by the addition of grit, sand, or perlite to the compost mix.
Low temperatures will slow growth. Arabidopsis will grow quite satisfactorily in continuous light. Under these conditions it flowers slightly earlier, produces less leaves, and seed production is reduced. Proprietary brands of plant fertilizer can be used to feed Arabidopsis. Growth, maintenance, and use of Arabidopsis genetic resources Protocol 2. Prepare a compost mix of six parts Levingtons M3: Each Vacupot tray takes 2 litres of compost mix.
Fill each pot and allow to settle by giving the pots a sharp tap and then compress the compost very lightly using finger tips. Place more compost mix on top until the tray is full. Using the riddle, sieve compost onto the top of the pots to give a fine bed on which to sow the seeds. Place the tray in water to soak, until the surface of the compost is damp, i. Sow the seed onto the surface of the compost. Sow seeds individually with a wetted microspatula or the fused end of a glass capillary tube. It is possible to scatter seeds by distributing the seed from a folded card either alone or mixed with sand or agar.
Growth should be visible after five days. Plants may be grown to maturity under this regime. Faster cycling of the plants can be achieved by growth under continuous illumination. For DNA preparation, plants can be grown under short day conditions, i. After seven to ten days i. Place the plants on benching covered in capillary matting overlaid with horticultural perforated plastic sheetingc and water from underneath. Alternatively, Arabidopsis can be germinated and grown directly in the glasshouse.
For standard lines it is not necessary to supplement the growth medium, although higher yields of seed may be obtained if you do so. Standard proprietary plant foods can be used. This is true for many of the ecotypes found in the UK. In nature these populations would germinate in the autumn and then over winter as rosettes and flower in the following April, producing seed in the June or July. The other category of lines, the summer annuals that include the commonly used lines Landsberg erecta and Columbia do not require a cold treatment in order to flower. Methods to prevent crossing Arabidopsis is self-fertile, but cross-pollination can occur within a glasshouse environment.
In order to minimize cross-pollination, several strategies can be adopted. Good glasshouse hygiene will minimize the number of insects and hence decrease the chance of cross-pollination through insect vectors. Space the material fairly far apart 20 cm to prevent flowers from different lines coming into contact with one another. Stake the material to prevent lodging. Stakes can be made from commercially available garden wire and pea rings. An alternative to this system can be made from cutting down clear plastic drinks bottles.
Harvesting After a period of about three to four weeks in optimal conditions, siliques will start to form. On maturation the silique dehisces and shatters scattering its contents. Several measures can be taken to collect the seed. Cellophane bags 45 mm x mm which are custom made by the suppliers are placed over the plants as the first silique ripens and are secured at the bottom of the bag by the use of sticky tape.
The bags 18 1: Growth, maintenance, and use of Arabidopsis genetic resources allow the exchange of air and water and so condensation does not build up on the inside of the bag and the remaining siliques can ripen. Allow the plants to thoroughly dry out. Then cut the plant off at the base, within the harvesting bag, and gently rub it to free the seeds from the siliques.
Collect the seeds directly from the bags by cutting a small hole in the corner. This precludes the need to sieve the material and so reduces the risk of mixing or contaminating seed lines. Some sieving of the material may be required. Careful cleaning of the system is required between use. Plants grown in small pots can be placed on their side within bags. This includes autoclaving plants and soil material before disposal and the thorough sterilization of all equipment that will be reused.
If mixing the seed with sand, to aid even distribution, then use sufficient sand to give an even covering over the top of the soil surface e. In general, the best philosophy to adopt in pest management is that of prevention rather than cure. The best way to minimize infestation of any kind in the glasshouse or growth room is to maintain good hygienic practise as outlined above. Nevertheless, it is almost inevitable that at some time the glasshouse will become infested with some kind of Arabidopsis pest.
Table 1 provides some guidelines on how to treat the most common types of infection that affect Arabidopsis. Prior to sowing the seeds, the insecticide is applied as a compost drench following the manufacturer's instructions. The insecticide gives protection against aphid, scarid fly, and thrip infections. However, to prevent the development of resistant strains, the insecticide is used in a planned programme of pest control, which also includes the use of nicotine shreds. Fly strips are used to act as insect traps that give a useful indication of the kind and extent of a particular infection.
The use of these chemicals should only be carried out by personnel who have received the necessary training to handle these materials and the manufacturer's instructions should be followed at all times. Warning signs indicating the time and type of treatment should be clearly posted. Ideally, all pesticide and fungicide treatments should be applied during the evenings or at 19 Mary Anderson and Fiona Wilson Table 1. Symptoms and treatment of common Arabidopsis pests and diseases Infection Symptom Treatment Aphids greenfly Aphids massed on leaves and flowering stem.
Distorted leaves due to aphids feeding on young tissues. Thrips Leaves are mottled with silver or white flecks. Thrips can accumulate in the inflorescence and can cause fertilization to fail. Scarid mushroom fly or fungus gnat Plants lack vigour and leaves turn yellow, without visible infection above the soil. Colourless maggots can be observed amongst the root tissue, where they feed. Adult flies gather around the plant and can be caught on fly strips. Red spider mite Infections occur under dry and hot growth conditions. Leaves have a silvery speckled appearance and in severe cases plants will yellow and wilt.
Underside of the leaves has the appearance of being dusted with a white powder suspended from thin white silk strands. Whitefly Found on the underside of leaves, the adults are small white flies and the nymphs are pale green. Infestation results in plants lacking vigour, wilting, and death. Botrytis A grey 'fluffy' mould grows on aerial plant parts and may be accompanied by degradation of the plant material. Mildew White powdery patches develop on leaves and flowering stems. Severe infections can result in plant wilt and death. In many circumstances it is better to throw all infected material away as infections can be very destructive.
At the first sign of infection spray plants thoroughly with Supercarb systemic fungicide Bio , following the manufacturer's instructions. Repeat treatment two weeks later. Growth, maintenance, and use of Arabidopsis genetic resources weekends. Areas that are undergoing treatment should be locked to prevent accidental exposure of personnel. Glasshouses and growth rooms should be thoroughly ventilated following treatment to flush with fresh air before personnel are allowed to re-enter the areas. Seed storage The most important factors that influence seed longevity are the temperature at which the seed is stored and the moisture content of the seed.
Generally, the higher either of these values, the shorter the shelf-life of the seed. In order to maximize seed viability prompt post-harvest treatment is required. How a seed is treated will greatly influence how long it can be stored. Seeds can remain viable for two years if stored in a dry atmosphere at room temperature.
Store the seeds in negative bags Kenro Ltd. Initial seed longevity studies applying the equations for the prediction of seed longevity 57 and following Harrington's 'rule of thumb' 58 suggest that these conditions will increase the longevity of the seeds from two to three years, at ambient conditions, to approximately 30 years. Examples of the time taken for different seed lots to reach low seed moisture contents are shown in Table 2. Determination of seed moisture content is described in Protocol 3.
Such freeze-thaw cycles should be kept to a minimum. Containers should be allowed to come to room temperature before opening to prevent the accumulation of condensation on the seeds and in the container. If condensation does accumulate, then the container should be allowed to thoroughly air dry before returning it to the freezer. Performing a quick germination test on a proportion of the harvested seed can be used to check seed viability.
Coated paper bags used to hold photograph negatives Kenro Ltd. The range of values is derived from the results of tests of three different seed lines placed in the environment control chamber at the same time. Germination tests can be performed by germinating the seed on perlite see Protocol 4 , agar see Protocol 5 , or wetted filter paper. Give the seeds a short cold treatment two to four days to help break any dormancy prior to placing them in the growth room. Determination of moisture content: Weigh a light-weight oven proof dish plus cover to four decimal places.
Record the weight VV1. Alternatively fold a small piece of aluminium foil to form a 2 x 3 cm packet and weigh this. Add mg of seeds to the dish or packet and weigh again W2. Prepare other replicates or different samples in the same way. Growth, maintenance, and use of Arabidopsis genetic resources 6.
Remove the dish or packet from the oven, replace the lid of the dish or seal the foil packet, and immediately place in a desiccator at room temperature to cool for min so the seeds can cool without absorbing water. Weigh each dish or packet W3. Do not leave the desiccator lid open during each weighing.
Calculate the moisture content as a percentage loss in weight using the following formula. Growing Arabidopsis with specific growth requirements Certain Arabidopsis mutants have specific growth requirements since they lack or have reduced sensitivity to growth regulators, or lack genes in specific metabolic pathways. Such lines can be germinated on Petri dishes see Protocol 4 where their phenotype can be verified and then transferred to soil for growing to maturity.
Half fill a 9 cm diameter Petri dish with sterile perlite. Wet the perlite with 1 mM KN03, plus the appropriate growth regulator see Table 3 , or with Gamborgs B5, until the perlite has absorbed the liquid about ml but there is no free liquid when the dish is tipped. Sow the seeds individually onto the surface of the perlite using a wetted microspatula, or the fused end of a glass capillary tube or Pasteur pipette. Allow the plants to germinate in the standard growth room conditions outlined in Protocol 1, 2.
When two to four true leaves have formed transplant the material to compost by gently scooping underneath the seedling with a microspatula, taking care not to damage the roots. Some carry over of the perlite will occur. Place the seedling in the soil and gently firm the soil around the transplanted material.
Maintain high humidity by placing the plants in a closed propagator for a few days to prevent excess water loss and to allow the plants to acclimatize. Plants can then be grown onto maturity as described in Protocol 2 whilst applying the appropriate growth regulator. Nutrient requirements of suspension cultures of soybean root cells. Sterile culture of Arabidopsis For some mutants with a very weak growth habit it may be necessary to germinate and establish the plants in tissue culture, then transplant them to soil when the plants have reached the four leaf to small rosette stage.
For these plants it is best to also surface sterilize the seed, as described in Protocol 5, to remove any bacteria or fungi which could have an inhibitory effect on plant growth. Alternatively, these plants may be started on sterile perlite plates see Protocol 4. To observe and screen plants with phenotypes expressed at the seedling stage e. If the plants are subsequently to be grown to maturity it is better to use the sterile culture 24 1: Growth, maintenance, and use of Arabidopsis genetic resources procedure described in Protocol 5, however if the seedlings will be discarded after scoring then a more basic agar medium can be substituted for the culture medium e.
In either case it is necessary to use seed that has been pre-sterilized as described in Protocol 5. Preparation of tissue culture medium 1. Weigh out the culture medium components according to Table 4 and dissolve in the appropriate volume of distilled water. Correct the pH of this solution to 5. Replace the top section of each Magenta pot. After autoclaving it may be necessary to place the Magenta pots in a sterile laminar flow cabinet with the two parts open slightly to allow any condensation to evaporate.
Alternatively the appropriate amount of agar can be added to the solution prepared in step 1 and autoclaved before pouring into presterilized Petri dishes. Sterilization and sowing of seed 1. Wrap the seed in a 9 cm filter paper disc and place it in a 30 ml Universal container. Shake the container gently for 10 min.
Pour out the bleach solution and replace it with 20 ml of sterile distilled water. Shake the container for approx. Repeat step 4 five more times. Remove the filter paper from the tube and unwrap it using sterile technique work in a laminar flow hood and use sterile forceps to unwrap the seeds on a sterile Petri dish.
Allow the seeds to dry or sow immediately. Use a sterile bacteriological loop, or fused Pasteur pipette, to pick up individual seeds for transfer to the culture medium in Petri dishes or Magenta pots. Propagation of plants in tissue culture 1. For short-term propagation e. For longer-term propagation seeds can be sown in Magenta pots at a density of five per pot, for growth to the small rosette stage, or one per pot if the plant is to be grown to maturity.
A revised medium for rapid growth and bioassays with tobacco tissue cultures. Growth, maintenance, and use of Arabidopsis genetic resources 4. In Life sciences research in space ed. In Cell genetics in higher plants, p. Plant Cell, 3, Plant Cell, 4, Plant Cell, 6, Plant Cell, 7, Clarke, M, Wei, W. Plant Cell, 9, Strategies and applications ed. In Methods in Arabidopsis research ed. Plant Cell, 5, International Seed Testing Association. In addition to DNA resources, the ABRC shares responsibility for seed stock preservation and distribution and dissemination of information for the Arabidopsis community.
Hence the DNA laboratory is integrated with the centre's other services, allowing these complementary efforts to be fully co-ordinated. While preservation of biological resources including DNA is a primary function of the stock centres, they also strive to serve the rapidly changing needs of plant molecular biology research. Protocols utilized for clone characterization, preservation, and dissemination will be featured. Other topics which will be discussed are the philosophy of DNA centre operation, organization of information relating to DNA stocks, and the future of DNA centre operations.
Missions of a plant DNA resource centre To provide services in the areas described above, a DNA centre must hold diverse and numerous stocks, maintain all of these without risk of loss, and be able to send samples of any of these items on very short notice. This must be achieved at minimal cost to the supporting research community, and pertinent information about the stocks must be maintained and distributed to the patrons of the centre.
The items to be maintained and the relative demand for these evolve rapidly. Hence flexibility of operation and close contact with the research community is imperative. Randy Scholl et al. The types of stocks currently held by ABRC include the following: We will outline the preservation, distribution, and handling issues as well as current techniques associated with each of these stocks.
Preservation of stocks Protocols for long-term storage of DNA stocks are well-tested and reliable for many vector types 1, 2. The stock centre context requires multiple back-ups for all stocks, similar to those devised for germplasm collections 3 , assured stable storage conditions, easy sampling and manipulation of the stocks. These criteria are all considered when methods of handling are chosen by ABRC. In some cases, more than one choice is available.
We will discuss the alternatives where appropriate. Hence, the treatment of genomic and cDNA clones is similar. Likewise the ESTs, which are usually received as individual bacterial strains containing plasmids, do not pose unique problems, as they are deposited in standard vectors 4, 5.
However, the rate at which ESTs are generated, donated, and ordered requires very careful consideration of all aspects of their handling, and procedures are modified whenever possible to simplify the handling and storage protocols. Many preservation methods are available for plasmids 6. Ultra-cold storage of the plasmid within the host bacterial cells is the most convenient method. Lyophilization of cells containing plasmids is also possible, as is cold storage of the dry plasmid DNA. The former method is 30 2: Preservation and handling of stock centre clones presently utilized by ABRC.
Back-up stocks of dried DNA are employed to ensure that stocks will not be lost. Lyophilization is not currently employed due to the initial start-up costs, effort, and uncertainty regarding long-range storage effects. To ensure that the strains containing specific clones are not lost, multiple samples are stored in ultra-low freezers that can be automatically transferred to back-up electrical power. Well-established techniques are employed in the handling of plasmid clones. It is current centre policy to request a sample of isolated DNA from the donor in addition to a live culture.
This greatly facilitates our ability to make the stock available quickly and ensure its safe preservation. The ABRC's policy is to receive the ESTs as frozen glycerol stocks and utilize these stocks as the sole in-house representatives of the clones. This maximizes storage redundancy while avoiding the problems associated with reculturing and establishment of fresh stocks. The effort required to deposit the stocks into replicated storage is minimized, and the errors which might arise during subculturing are avoided. Numerous cultures can be made for shipment from each working stock.
Some stocks present few problems in any of these areas, and others pose difficulties in all areas.
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Most ABRC stocks are maintained as live cultures and shipped as agar stabs from these cultures. The stocks represent starting points for experiments by users, are inexpensive and not overly labour-intensive. Hence, this method has been used extensively as a vehicle for stock distribution. The protocols for reestablishing user storage cultures from these are well defined.
Individual stab cultures can be sent to users by regular mail and do not require cold shipping. Cosmid libraries are usually handled in bulk or as subdivided samples see Chapters 3, 8, and 9 7. However, the large numbers of cosmid clones associated with physical and genetic map construction are maintained as individual clones. These are stored as glycerol stocks in tubes, with restriction fingerprint data collected for typing purposes. For individual cosmids, live working and back-up cultures are stored as glycerol stocks with an additional dry DNA back-up stock, just as are plasmids.
However, very clearly defined modifications of the handling procedures must be adhered to so that full inserts are retained 1, 9. Streak cultures on LB medium with the appropriate antibiotic. Pick five to ten preferably ten small individual colonies and isolate cosmid DNA using standard alkaline lysis 6. Digest isolated DNA with a restriction enzyme. Conduct electrophoresis on digested DNA on 0. The resulting band pattern should be compared to those published in original papers or the AIMS database http: Typically, the full-sized 32 2: Preservation and handling of stock centre clones Goodman laboratory cosmids result in five to ten bands when digested with restriction enzyme having a six base recognition sequence.
Colonies that grow larger usually have deletions and may be unsuitable as probes for the intended genomic regions. Storage methods include liquid buffer suspensions held at refrigerator temperatures, glycerol stocks held at ultra-low freezer temperatures, or freeze-dried stocks 1, 6. Any of these methods are convenient, with only the latter requiring any specialized equipment.
Since the aqueous stocks are relatively stable, easy and inexpensive to ship, and easy to aliquot, they are used for most ABRC libraries. Libraries received as glycerol stocks have also been maintained as such, with much success. Either of these methods results in stable stocks, which are easy to prepare and viable in storage. Care is exercised to assure that libraries are not amplified more times than necessary and that amplification procedures are unbiased. The general policy is that libraries are not accepted if they have been amplified more than twice prior to receipt. Procedures for screening phage libraries, amplifying phage libraries, and isolating phage DNA are well established 1, 6.
They are used for physical mapping and positional cloning, thus maintaining the integrity of these clones is imperative. Several YAC libraries have been received and are distributed Fortunately, the genome complexity of Arabidopsis is such that a few hundred YAC clones span the genome with high probability , 17 , and it is not necessary to resort to strategies such as pooled storage of clones. Working stocks of Arabidopsis YAC libraries are maintained as individual colonies gridded on plates which are stored, replicated, and then distributed in this form 14, The protocols for replenishing and storing such libraries are straightforward, but require care and some specialized equipment.
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Storage also requires ultra-low temperatures, unless freeze-drying is employed. Replicate library copies onto -URA microtitre plates. The plates are used as a working stock to obtain individual YAC clones or to make filter sets for hybridization. If an entire library is sent, shipping costs are charged to the receiver since this requires use of express carriers. Protocol 3 outlines a procedure for generating colony replicas in agar plugs which can be used for isolation of the artificial chromosome.
A procedure for isolation of DNA from the yeast cultures for hybridization is given in Protocol 4, and a high density colony hybridization procedure from a library plate is also described Protocol 5. Plug mould Bio-Rad Corp. Inoculate 25 ml -URA broth in 50 ml disposable tubes with the yeast clone.
A very dense culture is required. The caps must be loose to allow adequate aeration and it is recommended to tape the lids onto the tubes. Harvest cells by centrifuging at g, for 10 min. Centrifuge for 10 min at g. Pour off the supernatant and resuspend in Remove ul to a microcentrifuge tube to be used for a miniprep. Transfer the resuspended solution to an 1. The following set of manipulations is conducted using a plug mould step If a plug mould is unavailable, a 1 ml syringe can be substituted, as outlined in step At this point you should see release of pigment into buffer.
This indicates efficient lysis. Wrap Parafilm over the opening, and place on ice. This indicates that the lysis has worked. Pour off the buffer, allow the plug to come to the edge of the tube. Place the opening of the syringe on the top of the plug, pull back the plunger, and the plug will flow back into the syringe.
Avoid air bubbles in the syringe, since they will cause a break in the plug. Use the cells saved from the yeast plug preparation, or use fresh two to three day yeast culture. Centrifuge cells in a microcentrifuge at r. Resuspend the cells in ul of 0. Centrifuge spheroplasts for 10 sec in a microcentrifuge and resuspend in ul TE pH 8. Disperse cells by gentle pipetting.
Centrifuge for 15 min to pellet precipitate and transfer supernatant to a new tube. If the yeast cells had pigmentation the supernatant 36 2: Preservation and handling of stock centre clones should be coloured. If the supernatant is not coloured the proteolytic lysis of the cell walls was efficient and the yield of genomic DNA will be low.
Add an equal volume of ice-cold ethanol. Centrifuge for 15 min, take off supernatant, and dry down in Speed Vac or air dry Resuspend in ul of TE pH 8. Check the DNA by electrophoresis on an 0. Suggested loading, 30 ul for one genomic Southern. In fact, when cultures of the library members are pooled in an appropriate combinatorial fashion, PCR amplification on a finite set of isolated DNAs can implicate the single clone carrying an insertion of interest. The CIC library consists of twelve well microtitre plates, representing approximately five genome equivalents.
Hence, the probability that a clone containing a specific gene exists in the library is high, and its identification can be achieved with minimal effort via PCR. The CIC, three-dimensional pooling is as follows. First, the library is divided into 'superpools' within which the pooling is conducted. A superpool represents a set of clones small enough that the probability of finding at least one clone is less than 0. This is necessary in that a single positive clone is identified uniquely by the screening process, but multiple clones result in exponentially increasing numbers of non-positive clones being implicated by the screening process.
Consequently, there are four superpools i. Within the three plates, the dimensions are 'rows' the actual rows of the plates , 'columns' also columns of the plates , and 'half-plates'. Hence, within the three plates of a superpool, there are six half-plates, eight rows, and six columns the first and seventh, second and ninth columns, etc. Using this approach, a specific clone can be identified utilizing 24 PCR reactions. The centre has received the cells of this library, pooled as described above, from R.
The cells are maintained in liquid culture and are sent as a total of 80 samples. DNA can be isolated from these cells directly, or the cultures can be grown to obtain samples from which DNA can be isolated to allow large numbers of primer pairs to be screened against the population DNA can be prepared from the cultures using a scaled up method from Matallana et al. Some added effort is required of the centre to produce filters suitable for hybridization, but this is offset by the opportunity to have this critical step conducted under controlled conditions and to generate multiple filters from a few plates see Figure 1.
These filters are randomly tested to ensure quality. Hence, users unacquainted with yeast culture technology can simply hybridize three nylon filters to locate colonies of interest and thus do not have to establish a myriad of procedures. For procedures for the preparation and hybridization of YAC filters, see Chapter 8. Both P1 and BAC libraries accommodate large inserts approximately bp, and potentially larger and are maintained in E. These libraries are maintained as gridded sets of individual colonies in glycerol-containing medium on microtitre plates.
They are normally distributed as filters ready for hybridization. Similar hybridization protocols can be used for these as for YAC filters see Chapter 8 or ref. When necessary, the entire library can be provided. Individual clones of these libraries are distributed in the form of culture stabs, as a follow-up to filter probing. Hence, the procedures for handling these cultures are similar to those for plasmid and cosmid clones. The simplicity of DNA isolation and insert recovery for both P1 and BAC clones makes these highly attractive as vehicles for contig construction for positional cloning.
However, Liu personal communication recently found that the copy number 38 2: Preservation and handling of stock centre clones gure 1. Layout of BAG filters distributed for hybridization by users. Note that the filter is the size of four plates but that colonies of eight plates are superimposed on each of these four areas. Further, each plate position is spotted twice in a specific design noted as ' 4 x 4 pattern' on the figure. Hence each colony from a total of 32 plates can be unambiguously identified on the single filter hybridization. Even though the plasmid copy number is not amplified, a moderate amount 0.
The modified alkaline procedure outlined in Protocol 5 was developed for P1 clones 18, This procedure should be applicable for BAC clones as well. It is designed for mini-preparation from 3 ml of culture, although DNA isolations can be scaled up proportionately if necessary. For larger scale preparations commercially available isolation kits such as the one from Qiagen may be employed. Cultures can be inoculated directly from storage stocks, but this often results in lower DNA yields or potential amplification of contaminating cells.
Longer culturing is not recommended. Collect cells from 3 ml of culture into one 1. Preservation and handling of stock centre clones r. Remove any medium with a micropipette. Resuspend the cells in ul solution I about ul solution I for every 1 ml of culture by vortexing. Add 10 ul of lysozyme solution and mix. Add ul of solution II. Store the tubes on ice or at room temperature for min, but no longer.
Vortexing will shear high molecular weight DNA, not only fragmenting the clone molecules, but increasing the levels of host chromosomal DNA contamination, as well. Add ul of solution III. Mix by gently inverting the tubes ten times. Store the tubes on ice for min. Transfer the supernatant to fresh tubes.
Centrifuge at maximum speed for 5 min. Air dry, avoiding over-drying. Although the DNA can be used directly for various enzymatic manipulations, the following steps are recommended to increase DNA purity and improve restriction by more finicky enzymes. To each sample prepared from 3 ml of culture, add TE to a final volume of ul or more. Insufficient dilution will lead to coprecipitation of impurities in the following steps. If either DNA yield or impurity levels are unusually high, more dilution is called for. Extract again with phenol: Shearing of DNA is recommended before use in random prime probe preparation.
This includes the identification of proteinprotein interactions of Arabidopsis. The cloning system which identifies the interaction of the translation products of members of a eukaryotic cDNA library with the product of a clone of interest by the expression of both in yeast in conjunction with an interaction assay was perfected only in Its use has been immediately adopted in all model eukaryotic organisms, including Arabidopsis. Since the basic library components are theoretically useful for identifications of any protein-protein interactions, one such library can be shared by a large number of users of a species.
Hence, the ABRC has received and is distributing several two-hybrid libraries. For the yeast two-hybrid system, the basic cDNA expression library is cloned into a lambda or plasmid expression vector; in the former case the insert is excised as a plasmid prior to transformation into yeast. These libraries are distributed either as phage suspensions from which the users excise plasmid in bacterial colonies from or as previously excised plasmid DNA to be used for direct transformation of yeast. The system for expressing cDNA clones in yeast and identifying pairs of interacting proteins utilizes several yeast strains including one carrying the transformed cDNA library.
This technique was initially developed by Durfee and co-workers The currently distributed two-hybrid expression libraries 26 were supplied by the Walker and Theologis laboratories.
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Various modifications of techniques are in use for two-hybrid screening. The literature for specific libraries should be consulted for handling and strategies employed with a particular resource. Verification of stock identity and purity Clone identity and purity must be rigidly maintained and documented. Adherence to established microbial genetic practice largely accomplishes the 42 2: Preservation ana nananng of Stock centre clones required degree of control.
Verification and checking by restriction analysis and occasional hybridization experiments constitute the main checks. Documentation of the results by photography represents an effective means of record keeping. Individual donated plasmid clones are always streaked on appropriate selective medium, single resistant colonies picked, and separate minipreps grown and analysed by restriction analysis. A new culture, which will represent the ABRC stock, is established from a single colony having the correct restriction pattern and antibiotic phenotype.
Parallel procedures are employed for bacteriophage clones and cosmids. Failure will be assured if cosmid clones are handled improperly; particularly since the vectors for many of the established Arabidopsis cosmid vectors delete at high rates, and the colonies having the full insert grow very slowly. In this case, the very smallest colonies must be selected in the first step, and checked carefully prior to use of the stock as a probe.
Whenever a clone is received from the centre, it is important that the investigator carefully check the clone prior to use to avoid wasted effort 1, 6. The number of single clones picked for analysis varies for the type of stock. Two plasmid clones or phage plaques are usually sufficient. Adherence of the end-user to microbial practice and verification is important for ESTs in that these are not checked by the centre, due to the very large size of the current collection over 25 ESTs , it is highly recommended that EST clones be sequenced to confirm the identity prior to use.
Picking of up to ten colonies is recommended for cosmid clones. In most cases quality control checks of phage libraries do not require large efforts. With modern technology, phage analysis is reasonably convenient. Enough phage DNA for analysis ug can be recovered from 30 ml liquid lysates by polyethylene glycol precipitation. Other procedures are carried out according to standard manuals 6. Yeast artificial chromosome libraries are maintained as sets of individual clones, but these clones cannot be checked individually for correctness.
However, some quality control is possible by preparation of filters blotted to the culture plates and hybridization of these to several known Arabidopsis probes. The location of hybridizing colonies is checked for each library, and this constitutes a reasonable statistical check. The integrity of the libraries is evaluated using hybridization against plastid, repetitive DNA, and ribosomal repeat sequences. In addition, all YAC libraries are maintained under appropriate biosynthetic selection, and released from selection only in the growth cycle immediately preceding a hybridization experiment.
Genomic and cDNA libraries, which are maintained as bulk stocks, are not characterized extensively by the centre. Such libraries will typically be plated and hybridized with a few clones as a minimal check for representation. This can done in Arabidopsis by one of several approaches. These primer pairs theoretically only produce a PCR-generated band when a T-DNA insertion has occurred within or near the gene of interest, so that the two primer sites are opposed and a unique amplification product arises from PCR utilizing the primer pair.
On this basis, the insertion line can be identified by simple PCR and hybridization techniques. The associated pools are obtained. Plants can be isolated from the pools and grown to evaluate the phenotypic effect of the insertion. This approach has been utilized to isolate a number of gene-specific, T-DNA insertion mutant lines. Clearly, the task of locating an insertion occurring in a short genomic segment from a population of randomly generated T-DNA lines could be daunting. While screening the lines individually for insertion in a gene of interest is not feasible, PCR is sensitive enough that a single insertion line having opposed genomic T-DNA priming sites can be identified even if its DNA is pooled with as many as additional non-positive lines.
If DNA isolation and pooling is conducted in a strategic fashion, the process of sorting through a population to locate the line of interest can also be conducted efficiently and lends itself well to the distribution facilities already existing at the resource centre. The pooling strategy associated with these lines is as follows: Hence, each line is represented in one of ten row pools of and one of ten column pools of The screening of these 20 DNA samples, following identification of a positive PCR in a superpool, locates the positive insertion to a single set of ten lines.
Preservation and handling of stock centre clones experimenter to identify and isolate the desired line. Based on this strategy, when users request this stock, pools of are sent initially, the row-column DNAs are sent as follow-up for appropriate superpools, and then seeds of a single set of ten lines is sent following each successful analysis of the pools of The entire distribution process then becomes a simple three-step procedure.
The protocol used for isolating plant genomic DNA from pooled lines 25 is described in Protocol 6. This protocol is for 10 g of frozen tissue using ml Nalgene bottles. Swirl a few times during incubation and open slightly to release pressure. Remove bottles from the water-bath and let cool. This is very important—care is needed to add chloroform slowly to hot solution. Take the precaution to wear safety glasses and gloves whenever working with chloroform. Centrifuge for 10 min at 10 r. Remove the bottles from the centrifuge, taking care not to mix the phases.
Immediately remove the aqueous top phase into new Nalgene bottles, using a pipette. This step is to be done as quickly as possible.
Continued step 5, pipette the top layer into a Corex tube. When pipetting the aqueous phase, be sure not to carry over any debris or chloroform. If the solution is still greenish or not clear, repeat the chloroform extraction step. The solution will become cloudy after adding the CTAB, but will clear after several minutes.
Add additional ddW if no loose sediment is seen. If not, centrifuge for another 10 min. Decant the supernatant and add 4 ml 8 ml total for g tissue samples of 1 M NaCI. Transfer to smaller tubes 30 ml Corex, or 50 ml disposable centrifuge tubes. Centrifuge for 6 min at g. Be sure the pellet sticks firmly to the tube before decanting.
Decant the supernatant and let the pellet partially air dry or vacuum oven dry. The pellet should not dry completely otherwise it will not dissolve. Dissolve the pellet in ul 3 ml for g of tissue of TE, depending on the size of the pellet, overnight at room temperature. PCR can be somewhat problematic for untested primers and substrates. If difficulties arise the control primers can be applied to the appropriate control DNA pool of the kit.
Arabidopsis : a practical approach / edited by Zoe A. Wilson. - Version details - Trove
Otherwise, it is suggested that the pool DNA can be conserved by omitting the second step. The control primers for the CD pools are for act2 2 and this mutant is included in pool 1 of CD Preservation and handling of stock centre clones included control right border and ACT2 primers. The positive act2-l line of the control T-DNA pool possesses right borders on each side of the insert 2 , so primers homologous to the right border are necessary.
The amplification procedure is described in Protocol 7 24, It should be strongly emphasized that PCR amplification be followed-up with Southern hybridization to authenticate potential positive products, since border-border bands are extremely common. It is also very useful to employ negative PCR controls such as PCR reactions with each single primer employed in the experiments.
A small sample of a putative positive band can also be reamplified, subcloned, and sequenced for verification. The high-fidelity versions of the enzyme are preferable. Less can be used, although it is advisable to utilize higher quantities for the border primer. Note that for pools of lines, 30 cycles are usually sufficient, but 45 cycles may be required for pools of Add mineral oil if necessary.
PCR annealing step temperatures should be set to optimize conditions for the specific primer of each PCR primer pair; lower temperatures result in amplification of complicating nonspecific bands. Organization of stock information The data associated with DNA stocks are varied, complex, rapidly evolving, and interrelated.
Hence, data management is a complex issue. Stock centres do not serve as the primary organizational point for all such data, but must have access to these data, and must provide detailed information about clones to researchers. First the stock collection process is designed so that stock donors provide detailed data at the time of donation, and secondly, creating electronic access to the summarized data so that users have maximum use of the information. Donation forms asking for all pertinent information in an organized format are vital as is the identification of publications relating to the stocks.
Users are asked to provide all donation information in electronic form, whenever possible. Nucleotide sequence and related information such as homology search results should be accessible.
Some data must be maintained at the stock centre while others, such as sequence data, may best be organized elsewhere —specifically at the large sequence databases dedicated to providing this service. Even with ready access to databases, a local database maintained on either a microcomputer or a workstation is required for the organization of stock centre data and provides additional back-ups to ensure data is not lost. Allocation of significant fractions of the centre's personnel time is required for data management. The necessity to communicate with the researchers allied to the centre, and in some cases with specific sub-groups of the users, requires the careful organization and rapid mobilization.
A number of microcomputer programs are very efficient for these tasks. The extent to which these lists are made public are at the discretion of each centre. The future It is clear that demand for DNA stocks will continue to increase rapidly in the near future and that the number and complexity of stocks with which stock centres will have to deal will also increase. In some cases, adding new types of stocks will mean greater need for financial support, but the labour and technical demands may actually be reduced by others. The recent development of 48 2: Preservation and handling of stock centre clones various types of large-insert libraries which are easier to use is a good example.
Also, robotics is allowing the centre to provide additional services. A physical map of Arabidopsis is now essentially complete 27, BAC and P1 clones are easier than are YACs for the user to manipulate, although the insert sizes are currently smaller. Hence, distribution of these as either filter sets, pooled DNAs, or grids of clones require automated handling. The primary solution to the distribution problems posed by these resources may be to distribute the full library only as filters for hybridization, with individual clones from the library available for direct shipping.
In conclusion, the ABRC has established an extensive collection of DNA and seed stock items and has sent over samples to researchers throughout the world during the first six years of stock distribution. The centre has already moved several steps beyond its initially envisioned scope by acquiring the large collection of ESTs, large-insert libraries, and isolated DNA for PCR screening. The next foreseeable steps will involve organized sets of mutants e.
It is obvious that similar sudden, unanticipated reorientations will be necessary in the future. The challenge will be to serve researchers with divergent needs while maintaining reasonable economy of operation. Current protocols in molecular biology. Wiley Interscience, New York. In Cryopreservation of plant cells and organs ed. Plant Physiol, , Plant Cell, 1, Plant Biol, 11, In Methods in molecular biology: DNA Research, 5, Introduction New markers can be positioned on a genetic or molecular map by comparison of the segregation of parental alleles in a population containing recombinant chromosomes, with the segregation data of other markers in the same population.
Marker order is determined by identifying which markers e. The fewer the recombination events that separate two markers the closer together they map on a chromosome. If one recombination event separates two markers in chromosomes, then the two markers are considered to map 1 centimorgan cM apart. The central requirement for genetic mapping is therefore the ability to distinguish the parental alleles at each locus. Phenotypic markers were the basis of the original genetic maps. The first molecular markers were restriction fragment length polymorphism RFLP markers 1 see Section 3.
Arabidopsis : a practical approach
These are genomic DNA fragments that show a difference in size between restriction digests of parental DNA, as assayed using Southern blot hybridization, with a particular probe. RFLP markers are generally low-copy sequences and have the advantage over phenotypic markers in that they are co-dominant, i. More recently, a number of polymerase chain reaction PCR -based markers have been developed that significantly speed-up the mapping procedure see Section 3.
The major disadvantage of using F3 families for mapping is that when the F3 seed are exhausted new populations must be generated and the segregation of markers re-analysed to produce a new map. Clare Lister et al. Schematic illustration of recombinant chromosomes. Three pairs of recombinant chromosomes A, B, and C are homozygous for all loci shown.
Arabidopsis
Black represents one genotype, white the other genotype. The composition of each recombinant chromosome is determined by analysis of the segregation of the RFLP markers at the seven loci i-vii. The order of the markers is determined by the position of recombination events. An alternative mapping population, used successfully in maize 4 , wheat 5 , and pea 6 mapping is a recombinant inbred RI population.
A large number of F2 plants are selfed and then one progeny F3 plant, chosen at random from each F2, is allowed to self and set seed. This process is repeated for sufficient generations until the lines are highly homozygous see Figure 2V. Theoretically, at the F8 generation only 0. RI lines have many advantages for mapping markers compared with F3 families: Since mapping still requires the identification of a polymorphism between the parental lines it is therefore advantageous to have multiple mapping populations, derived from different parents.
If a polymorphism is not detected between one set of parental ecotypes, it can often be detected between another. A number of A. These lines have been generated from different pairs of parental ecotypes. Two sets of lines, from crosses between the ecotypes Col-0 with Nd-1 and Wei with Ksk-1 were generated by Holub and Beynon 9. These RI lines were specifically developed to map fungal resistance genes where one parent is sensitive to a particular pathogen and the other is resistant , but may also be used for mapping with 52 3: Genetic mapping using recombinant inbred lines Figure2.
You also may like to try some of these bookshops , which may or may not sell this item. Separate different tags with a comma. To include a comma in your tag, surround the tag with double quotes. Skip to content Skip to search. Published Oxford ; New York: Oxford University Press, Language English View all editions Prev Next edition 2 of 2. Check copyright status Cite this Title Arabidopsis: Other Authors Wilson, Zoe A. Physical Description xix, p.
Subjects Arabidopsis -- Molecuar aspects -- Laboratory manuals. Arabidopsis -- Molecular aspects -- Laboratory manuals. Arabidopsis -- Laboratory manuals. Contents Machine derived contents note: List of Contributors Abbreviations 1. Greg Briarty, and Zoe A. Classical and molecular cytogenetics of Arabidopsis, G. Tissue culture, transformation, and transient gene expression in Arabidopsis, Keith Lindsey and Wenbin Wei 7. Aarts, Csaba Koncz, and Andy Pereira 8. Web-based bioinformatic tools for Arabidopsis researchers, Seung Y. Rhee and David J. Notes Includes bibliographical references and index.
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