Agrobacterium Transformation Kit
Contents
1. 9 Plate 20 ul of the recovered cells onto each selective Agrobacterium Medium Agar plate using Roll amp Grow Plating Beads 10 Invert plates and incubate at 28 30 C for 48 72 hours 6 Detailed Protocol 1 Prepare Agrobacterium Medium with the appropriate antibiotics Add the contents of the media pouch 16 5 g to 1 0 L of purified water and autoclave Allow the media to cool to 55 C before the addition of any antibiotics and bacterial inoculation See Sections 3 2 and 3 4 for a discussion of antibiotics selection and controls 2 Prepare Agrobacterium Medium Agar with the appropriate antibiotics Add the contents of the agar pouch 31 5 g to 1 0 L of purified water and autoclave Allow the media to cool to 55 C before the addition of any antibiotics and pour into plates See Section 3 2 and 3 4 for a discussion of antibiotics selection and controls 3 Inoculate prepared Agrobacterium Medium with a single Agrobacterium colony from a plate less than two weeks old Incubate at least overnight 18 38 hours at 28 30 C to an ODgoo of 1 0 2 0 in a shaker at 200 rpm Allow 5 ml of medium for each transformation reaction For example grow 25 ml of cul ture in order to perform 5 transformations NOTE For optimal and consistent growth bacteria should be grown in a sealed 50 ml conical tube with 20 ml of media Do not poke holes in the tube lid or otherwise allow aeration of the culture Do not grow cells above 32 C as transformation e2. If the highest possible transformation efficiency is desired follow the electroporation protocol and optimize your specific plasmid strain using a range of starting plasmid DNA quantities As a starting point Qbiogene recommends the use of 500 ng of plasmid DNA for each transformation reaction Successful transformation has been achieved with as little as 1 ng of plasmid with both the freeze thaw and electroporation protocols 3 6 Choosing an Agrobacterium Strain Many strains of A tumefaciens or A rhizogenes have been used to transform a variety of different plants and the most successful strain for a particular plant should be researched prior to using this kit While most strains in use for transformation will contain the genes necessary to move a gene of interest into a plant cell it can be determined whether or not a particular strain of Agrobacterium is phytopathogenic using a simple PCR test based on the virD2 and ipt genes 3 7 Suitability of Agrobacterium Mediated Transformation Although Agrobacterium transformation remains the most common mode of transformation of dicotyledo nous plants there remain many species or strains that are resistant to Agrobacterium infection There has been much advancement in determining a plant s ability to be transformed such as a polymerase chain reaction assay which identifies the presence of absence of an important allele H2A 1 Agrobacterium mediated transformation of monocotyledonous plants such a
3. 3 Electroporate the cells at a 1 5kV cm with a pulse of 5 msec 4 Transfer cells from the cuvette to a new 15 ml tube containing 0 9 ml of prepared Agrobacterium Medium 10 Incubate samples in a 28 30 C shaker at 200 rpm for 30 minutes to 3 hours NOTE For optimal results transformed cells should be allowed to recover for 3 hours In many cases how ever recovery of only 30 minutes can lead to a number of colonies that is sufficient for further growth and experimentation 11 Plate 20 ul of the recovered cells onto selective Agrobacterium Medium Agar plates using Roll amp Grow Plating Beads a Aspetically dispense 6 7 Roll amp Grow Plating Beads onto each agar plate b Replace the plate cover and gently roll the beads back and forth for approximately 30 seconds to dis perse the cells Multiple plates can be stacked and rolled simultaneously c Remove the lid and roll beads from the plate into a biohazard waste receptacle NOTE In most cases plating 20 ul of the transformation reaction will result in a number of well isolated colonies from which it is easy to calculate the transformation efficiency If there are too many colonies plate again using a smaller volume or dilution If there are few colonies plate a higher volume or spin down the 1 ml culture and remove most of the media prior to plating the entire cell pellet 12 Replace the lid invert the plates and incubate at 28 30 C for 48 72 hours NOTE tis normal for co
4. method does not yield a sufficient number of colonies The size of the construct being transformed can have a significant impact on the transformation efficiency Agrobacterium shuttle plasmids are typically large and the inserted DNA T DNA may contain the gene of interest its regulating sequences as well as an additional selection marker If a low number of transformed colonies appear after a 72 hour incubation it may be possible to increase the efficiency by repeating the transformation using more than 500 ng of plasmid DNA The purity and conformation of the plasmid DNA is important Plasmid DNA should be prepared carefully so as to ensure that contaminating RNA and other proteins are absent from the preparation and that the vast majority of recovered plasmid DNA is supercoiled Linearized DNA will not efficiently transform bacteria Ensure that the Agrobacterium culture was grown to an ODggg of 1 0 to 2 0 and that 5 ml of culture is used for each transformation Ensure that the correct antibiotics and or other selective agents were used that they were freshly prepared and that they were added at the correct concentrations Any Questions Call Technical Support at 800 424 6101 11 Agrobacterium Tra 8 Recommended Reference Format for Publication Agrobacterium species or strain was transformed using the Agrobacterium Transformation Kit Qbiogene Inc CA 9 References 1 Glick B R and J E Thomp
5. Qbiogene Application Manual Agrobacterium Transformation Kit Freeze Thaw Transformation or Electroporation of Plasmid DNA into Agrobacterium with a Single Solution Revision 3301 100 4F01 Q BlO gene Transformation Kit Agrobacterium Transformation Kit Freeze Thaw Transformation or Electroporation of Plasmid DNA into Agrobacterium with a Single Solution Application Manual Revision 3301 100 4F01 Catalog 3301 100 25 preps Storage Ambient temperature 15 30 C Any Questions Call Technical Support at 800 424 6101 3 1 introduction niacin a iia y pietia en pipa a Urs E ade 5 2 Kit Components and User Supplied Materials 5 2 1 Agrobacterium Transformation Kit Components eeene 5 2 2 User Supplied Materials ssssseeenemn nnns 5 3 Important Considerations before Use 0 cece eee eee eens 6 3 1 Electroporation or Freeze Thaw Transformation 6 Gie2 gt 3 CET 6 3 3 Growth Medium for Agrobacteri mM sisisi a 7 3 4 Appropriate Experimental Controls 7 34 Quamntity of Plasmid DNA aig eter entr rice cct rd ree cc n n n 3 6 Choosing an Agrobacterium Strain sss 7 3 7 Suitability of Agrobacterium Mediated Transformation 7 4 Safety PrecautioNs 22 55252225252 020058 a e ERR RR RR n 8 5 Short Protocol for Experienced Users 0 0 cee ccc e eee neces 8 6 Detailed Protocol 0 cc
6. ashige amp Skoog Basal Medium Salts Vitamins 10L Pouch 5100 125 Murashige amp Skoog Complete Medium Salts Vitamins Sucrose 1 L Pouch 5100 114 Murashige amp Skoog Complete Medium Salts Vitamins Sucrose 500 ml 5100 225 Murashige amp Skoog Complete Agar Salts Vitamins Sucrose Agar 1 L Pouch 5100 042 Murashige amp Skoog Vitamins Powder 5g 12 visit us on the web at www qbiogene com 1 Transformation Kit 5100 044 Murashige amp Skoog Vitamins 1000X 20 ml 5101 035 Gamborg s B5 Basal Salts 10 L Pouch 5101 014 Gamborg s B5 Basal Salts 10X 500 ml 5101 335 Gamborg s B5 Basal Medium Salts Vitamins 10 L Pouch 5101 125 Gamborg s B5 Complete Medium Salts Vitamins Sucrose 1 L Pouch 5101 114 Gamborg s B5 Complete Medium Salts Vitamins Sucrose 500 ml 5101 225 Gamborg s B5 Complete Agar Salts Vitamins Sucrose Agar 1 L Pouch 5101 042 Gamborg s Vitamins Powder 59 5101 044 Gamborg s Vitamins 1000X 20 ml 5102 035 Chu s N6 Basal Salts 10 L Pouch 5102 014 Chu s N6 Basal Salts 10X 500 ml 5102 335 Chu s N6 Basal Medium Salts Vitamins 10 L Pouch 5102 125 Chu s N6 Complete Medium Salts Vitamins Sucrose 1 L Pouch 5102 114 Chu s N6 Complete Medium Salts Vitamins Sucrose 500 ml 5102 225 Chu s N6 Complete Agar Salts Vitamins Sucrose Agar 1 L Pouch 5102 042 Chu s N6 Vitamins Powder 5g 5102 044 Chu s N6 Vitamins 1000X 20 ml Plating Tools amp Culture Dishes 5000 552 Roll amp Gr
7. ause irritation Wear personal protective equipment to prevent contact with the skin or mucus membranes gloves lab coat and eye protection Consult the enclosed Material Safety Data Sheet for additional details 5 Short Protocol for Experienced Users 1 Prepare Agrobacterium Medium and Agrobacterium Medium Agar plates with the appropriate antibiotics 2 Grow Agrobacterium 5 ml per transformation to an ODgoo of 1 0 2 0 in a 28 30 C shaker at 200 rpm 3 Aliquot 5 ml of culture into each sample tube 4 Centrifuge at 3 500 x g for 5 min at room temperature or 4 C and remove supernatant 5 Resuspend each 5 ml cell pellet in 90 ul of Agrobacterium Transformation Solution 6 Add 500 ng plasmid DNA and mix gently 7 Follow EITHER step 7a OR 7b below 7a Freeze Thaw Transformation 1 Submerge the portion of the sample tube containing the reagents into liquid nitrogen for 1 minute 2 Transfer sample to a 30 C water bath for 5 minutes 3 Add 0 9 ml of prepared Agrobacterium Medium 7b Electroporation 1 Transfer resuspended cells to a cold 1 mm cuvette 2 Wipe off all ice or condensation to reduce the potential for arching 8 visit us on the web at www qbiogene com ransformation Kit 3 Electroporate the cells at a 1 5kV cm with a pulse of 5 msec 4 Transfer cells from the cuvette to a new 15 ml tube containing 0 9 ml of prepared Agrobacterium Medium 8 Incubate samples in a 28 30 C shaker at 200 rpm for 30 minutes to 3 hours
8. c cece ccc eee IH 9 7 Troubleshooting jcc rrr hm nme 11 7 1 No Colonies on Agar Plate after Transformation 11 7 2 Lower than Expected Transformation Efficiency ssennn 11 8 Recommended Reference Format for Publication 12 9 References 2 545444 aded rd Rari ard aded dur aen 12 105 Related Products 55555555 13582350 EEna ER 4A sa Ria nidad 12 11 Product Use Limitation amp Warranty oooooooocccnoornnnnoo 14 4 visit us on the web at www qbiogene com Transformation Kit 1 Introduction Phytopathogenic Agrobacterium species are important tools for the study of plant genomics and proteomics Native to soil these organisms contain DNA sequences that confer virulence via the transfer of genes from the Agrobacterium to the plant cell Researchers can take advantage of this ability by inserting a gene of interest into a plasmid that is then transformed into an Agrobacterium containing a second plasmid with vir ulence genes a binary vector system Once exposed to susceptible plant tissue molecular signals lead to the transfer of the gene of interest into the plant cell Agrobacteria are difficult to work with in the lab so preliminary subcloning and clone confirmation usually takes place in coli Recombinant plasmid DNA is then purified from coli and the reagents in the Agrobacterium Transformation Kit are used to transform DNA into the Agrobacterium in three basi
9. c steps Agrobacteria are first grown in liquid medium next they are transformed using either a freeze thaw method or via electroporation Lastly transformed cells are plated onto selective agar plates and allowed to grow for 2 3 days into healthy colonies The Agrobacterium Transformation Kit contains a single transformation solution for use in either a freeze thaw or electroporation method Optimized Agrobacterium Medium and Agar are also included along with Roll amp Grow Plating Beads for easy spreading of transformed cells Once media preparation is com plete this easy transformation procedure takes less than 45 minutes with minimal hands on time 2 Kit Components and User Supplied Materials 2 1 Agrobacterium Transformation Kit Components Agrobacterium Transformation Solution 2 5 ml Agrobacterium Medium Pouch 16 5 g 1 each Agrobacterium Medium Agar Pouch 31 5 g 1 each Roll amp Grow Plating Beads 1 each User Manual 1 each MSDS 1 each Certificate of Analysis 1 each 2 2 User Supplied Materials Purified plasmid DNA from E coli culture 500 ng per transformation See section 3 5 Agrobacterium strains rhizogenes or tumefaciens 50 ml culture tubes with caps for growth of Agrobacterium Shaking incubator set to 28 30 C Antibiotics or other selection reagents Autoclave Centrifuge capable of handling gt 5 ml of culture at 3 500 x g Any Questions Call Technical Support at 800 424 6101 5 Agrobacteriu
10. fficiency can be lost Do not overgrow the culture If media has become visibly clear with clumps of cells the culture should not be used for transformation If more than 4 transformations gt 20 ml are planned use multiple 50 ml culture tubes If absolute consistency is required for your experiment pool the cell cultures together and mix gently before aliquoting into separate tubes in step 5 4 Place Agrobacterium Transformation Solution on ice If using the electroporation method place cuvettes on ice also 5 Aliquot 5 ml of Agrobacterium culture into each sample tube Any Questions Call Technical Support at 800 424 6101 9 Agrobacterium Tran 6 Centrifuge at 3 500 x g for 5 minutes at room temperature or 4 C and remove supernatant 7 Resuspend each 5 ml cell pellet in 90 ul of ice cold Agrobacterium Transformation Solution 8 Add 500 ng plasmid DNA and mix gently NOTE Some plasmids especially large constructs may require the use of more than 500 ng of DNA See section 3 5 for more information 9 Follow EITHER step 9a OR 9b below 9a Freeze Thaw Transformation 1 Submerge the portion of the sample tube containing the reagents into liquid nitrogen for 1 minute 2 Transfer sample to a 30 C water bath for 5 minutes 3 Add 0 9 ml of prepared Agrobacterium Medium 9b Electroporation 1 Transfer resuspended cells to a cold 1 mm cuvette 2 Wipe off all ice or condensation to reduce the potenetial for arching
11. lity of Qbiogene Inc hereunder shall be limited to at our dis cretion no replacement or compensation product credits refund of the purchase price of or the replace ment of materials that do not meet our specification By acceptance of the product Buyer indemnifies and holds Qbiogene Inc harmless against and assumes all liability for the consequence of its use or misuse by the Buyer its employees or others including but not limited to the cost of handling Said refund or replacement is conditioned on Buyer notifying Qbiogene Inc within thirty 30 days of receipt of product Failure of Buyer to give said notice within thirty 30 days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material s FastRNA FastDNA FastPrep RNase Erase Roll amp Grow and BIO 1019 Systems are registered trade marks of Qbiogene Inc PhoenIX and RapidPURE are trademarks of Qbiogene Inc mTRAP is a trademark of Active Motif GROWTEK M is a trademark of Bel Art Laboratories 14 visit us on the web at www gbiogene com NORTH AMERICA UK Phone 1 800 424 6101 or 760 929 1700 Phone 0800 328 8401 E mail technical gbiogene com E mail techservuk gbiogene com FRANCE GERMANY Phone 03 88 67 54 25 Phone 0800 409 0580 E mail techservfr gbiogene com E mail techservde gbiogene com www gbiogene com Q BlOgene
12. lonies to take several days to appear After this initial growth period the bacteria will return to a normal growth cycle allowing the use of an overnight media culture for downstream applications 10 visit us on the web at www gbiogene com Transformation Kit 7 Troubleshooting 7 1 No Colonies on Agar Plate after Transformation It can often take at least 72 hours for transformed Agrobacterium to form colonies on selective agar plates Ensure that the appropriate controls are used in order to monitor growth of non transformed Agrobacterium as well as those transformed with the plasmid DNA Ensure that the correct antibiotics and or other selective agents were used that they were freshly prepared and that they were added at the correct concentrations Transformation may have occurred at a lower efficiency than expected See the next section for more information 7 2 Lower than Expected Transformation Efficiency Transformation efficiency depends upon several critical factors and can vary from 102 to 107 as these fac tors change from experiment to experiment Most applications will not require the production of a large number of independent transformed colonies in theory only one colony should be used to inoculate a larger culture for the transformation of the plant itself In almost every case transformation via electroporation will be more efficient than the freeze thaw method and may be tried if the freeze thaw
13. m Tran For Electroporation Only Electroporator 1 mm cuvettes 15 ml conical tubes 2 per transformation For Freeze Thaw Transformation Only 15 ml conical tubes 1 per transformation Water bath or heat block set to 30 C Ice bucket Liquid nitrogen or an ethanol dry ice slurry deep enough to submerge gt 1ml of reagents 3 Important Considerations before Use 3 1 Electroporation vs Freeze Thaw Transformation The Agrobacterium Transformation Kit provides the protocols and reagents for either a freeze thaw or an electroporation transformation Electroporation is the most efficient process while freeze thaw transforma tion is a shorter protocol that does not require specialized equipment Depending on the downstream appli cation the freeze thaw protocol may give an adequate number of colonies in less time using only one tube Electroporation is recommended for high efficiency applications such as the construction of genomic or cDNA libraries in Agrobacterium and for the isolation of genes by complementation of plant mutants 3 2 Selection The selective markers in the strain being transformed and on the plasmid being used dictate what type of selective mediums to employ Many commercially available strains of Agrobacteria are antibiotic resistant and many commercially available plasmids confer antibiotic resistance or employ use of the lacZ operon to cleave B galactosidase Often there is a third selective marker introduced a
14. ow Plating Beads 2 tubes 5112 043 GROWTEK M Culture Vessel Fach 5110 033 Magenta Box GA7 Pack of 8 8 boxes 5110 053 Magenta Box GA7 5 packs of 8 Case of 40 5110 043 Magenta Box Tray 7 Way Each 5111 033 Petri Dishes Sterile Polystyrene 100 mm x 20 mm 20 plates 5111 053 Petri Dishes Sterile Polystyrene 100 mm x 20 mm 500 plates Genomic DNA and RNA Purification 6540 400 FastDNA Kit 100 preps 6560 200 FastDNA SPIN Kit for Soil 50 preps 6045 050 FastRNA Pro Green Kit Plants and Animals 50 preps 6070 050 FastRNA Pro Soil Direct Kit 50 preps 6075 050 FastRNA Pro Soil Indirect Kit 50 preps 6001 100 FastPrep FP100A Instrument 100V 1 6001 120 FastPrep FP120A Instrument 120V 1 6001 220 FastPrep FP220A Instrument 220V 1 1201 100 mTRAP Midi Kit 24 preps 1201 200 mTRAP Maxi Kit 6 preps 1201 300 mTRAP Total Kit 12 preps Any Questions Call Technical Support at 800 424 6101 13 Agrobacterium Tran 11 Product Use Limitation amp Warranty Unless otherwise indicated this product is for research use only Purchase of Qbiogene Inc products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties Qbiogene Inc makes no warranty of any kind expressed or implied including merchantability or fitness for any particular purpose except that the products sold will meet our specifications at the time of delivery Buyer s exclusive remedy and the sole liabi
15. s maize and rice is also becoming routine While the reagents in the Agrobacterium Transformation Kit should successfully allow plasmid transforma tion into either A tumefaciens or A rhizogenes species the ultimate transformability of plants with the gene of interest is beyond the scope of this kit Any Questions Call Technical Support at 800 424 6101 7 Agrobacterium Tran 4 Safety Precautions Some Agrobacterium strains are classified as plant pests and should be only be used after obtaining all rel evant state and federal permits and after following all appropriate guidelines for use and containment The United States Department of Agriculture USDA requires permits for the importation and interstate move ment of plant pathogens under the authority of 7 CFR 330 Plant pathogens include nematodes bacteria fungi viruses viroids phytoplasms or any organisms similar to or allied with any of the foregoing or any infectious substances which can directly or indirectly injure or cause disease or damage in any plants or parts thereof or any processed manufactured or other products of plants For more information in the United States contact the USDA www aphis usda gov ppq permits or your state department of agriculture For more information in other countries contact your local environmental or other appropriate authority Agrobacterium Transformation Solution contains components that when in contact with human tissue may c
16. s part of the insert DNA that will be transferred into the plant T DNA to confer selectability at that stage Antibiotics commonly used with Agrobacterium and their concentration ranges are below Antibiotic Concentration Ampicillin 20 100 ug ml Carbenicillin 500 1000 ug ml Chloramphenicol 3 25 ug ml Gentomycin 20 ug ml Kanamycin 20 100 pg ml Rifampin 30 ug ml Streptomycin 15 100 ug ml Sulfonamide 100 ug ml Tetracycline 5 10 ug ml 6 visit us on the web at www qbiogene com Transformation Kit 3 8 Growth Medium for Agrobacterium Qbiogene does not recommend the use of any growth medium other than the Agrobacterium Medium and Agrobacterium Medium Agar provided in the kit LB Medium as well as other versions of Agrobacterium media may increase growth and processing time and will lead to few if any transformants 3 4 Appropriate Experimental Controls Negative Control Competent bacteria without added plasmid should be plated on positive selection medium to confirm the prepared competent cells did not contain plasmid prior to use Positive Control Transform a preexisting plasmid at a known concentration to confirm cell competency check positive selection and permit calculation of transformation efficiency 3 5 Quantity of Plasmid DNA The amount of plasmid DNA added for each reaction plays an important role in the final transformation effi ciency Larger plasmids may need to be added at a higher quantity than smaller plasmids
17. son Methods in Plant Molecular Biology and Biotechnology CRC Press Inc Boca Raton Florida 1993 2 Nickoloff J A Electroporation Protocols for Microorganisms Humana Press Totowa New Jersey 1995 3 Haas J H Moore L W Ream W and S Manulis Applied and Environmental Microbiology 2879 2884 Aug 1995 4 Zhu et al Plant Physiology Jun 132 2 494 505 2003 5 McCormac A C Elliot M C and D F Chen Molecular Biotechnology 9 155 159 1998 10 Related Products Cat 4 Description Size Plasmid Purification 2075 400 PhoenIXTM Gigaprep Kit 5 preps 2075 300 PhoenlX Maxiprep Kit 25 preps 2075 350 PhoenIXTM Maxiprep Low Copy Refill Kit 12 preps 2075 600 PhoenIXTM Filter Maxiprep Kit 10 preps 2075 200 PhoenlX Midiprep Kit 25 preps 2075 250 PhoenlX Midiprep Low Copy Refill Kit 12 preps 2067 200 RapidPURETM Plasmid Mini Kit 60 preps 2067 200 RapidPURE Plasmid Mini 96 Kit 96 preps 2000 200 MiniPrep Express Matrix 1 250 preps Growth Media 3301 012 Agrobacterium Medium 227 g 0 5 Ib 3301 075 Agrobacterium Medium 10 x 1 L Pouches 3301 212 Agrobacterium Medium Agar 227 g 0 5 Ib 3301 275 Agrobacterium Medium Agar 10 x 1 L Pouches 5100 035 Murashige amp Skoog Basal Salts 10 L Pouch 5100 014 Murashige amp Skoog Basal Salts 10X 500 ml 5100 045 Murashige amp Skoog 1 2 Strength Basal Salts Arabidopsis Basal Salts 10 L Pouch 5103 035 Murashige amp Skoog Basal Salts with Gamborg s B5 Vitamins 10 L Pouch 5100 335 Mur
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