Transform & Grow™ Bacterial Transformation Kit
Contents
1. 19 12 Related Products cic aii ai err a wil 19 12 1 Plasmid Purification Kits ssssssseeenne 19 12 2 Other Transformation Kits and Tools ssssnme 19 12 3 Molecular Biology Certified M Growth Media 20 13 Product Use Limitation 21 Any Questions Call Technical Support at 800 424 6101 5 Transform Grow Bacterial T 1 Introduction Transformation is a common method of introducing foreign DNA into a bacterial host cell An essential part of the DNA cloning process bacterial transformation steps are required at many points during the progres sion of a molecular biology experiment Transformation is used to introduce plasmid ligation reactions into bacteria for clone replication Recombinant plasmids are transformed into different bacterial host cells for large scale replication and for maintenance and identification to express proteins to study mutagenized clones and to meet a wide variety of other research needs Bacterial transformation was first demonstrated by Mandel and Higa 1 who reported that bacteriophage DNA introduced into E coli cells produced infectious phage centers Cohen and colleagues subsequently demonstrated calcium dependent E coli transformation with plasmids carrying antibiotic resistance that resulted in the plasmid DNA being maintained in the cell as ind2. Quick Protocol for Experienced Users 10 7 Detailed Protocol uns na aan 11 7 1 Culture Preparation 11 1 2 Preparation of Competent Cells sss 12 7 3 Transformation of Plasmid DNA sse 13 4 visit us on the web at www qbiogene com ow Bacterial Transformation Kit 8 5 lt 14 8 1 Does Transformation Efficiency Vary with the Volume of Competent Cells PTOQUCSO tancia tte tet ite lan ia 14 8 2 Does Transformation Efficiency Vary with Plasmid Size 14 8 3 Do Plasmids Containing Different Antibiotic Resistance Genes Have Different Transformation Efficiencies sss 15 9 Troubleshooting gt remm n c c xc o s 16 9 1 Why don t have colonies on my supercoiled transformation plate 16 9 2 Why don t have colonies on my ligation transformation plate 17 9 3 have too many colonies or a bacterial lawn on my transformation plate 17 9 4 What if the colonies seem 100 large u nee 18 9 5 What if there are two or more types of 18 10 Recommended Reference Format for Publication 19 11 SREIBLENCES ra ace tee
3. This is not often a problem when an existing plasmid is trans formed among different strains when using nano or picograms of DNA It may become a problem during a ligation transformation if a high percentage of parent vector from the plasmid or the insert is present To Calculate Transformation Efficiency Total Cell Volume ul 1 of Colonies x Volume Plated ul ug transformed on Plate CFU ug Example 100 ul competent cells containing 5 ng 0 005 ug plasmid DNA was diluted into 900 ul SUPER COMP Media for 37 C incubation 100 ul was spread on the plate and 697 colonies were counted 697 x 1000 ul 1 6 100 ul 0 005 ug 1 39 x 10 transformations per ug DNA 5 Safety Precautions The Transform amp Grow Bacterial Transformation Kit contains components that when in contact with human tissue or during inhalation may cause irritation Wear personal protective equipment to prevent skin contact e g gloves lab coat and eye protection and prevent inhalation of reagent vapors and consump tion of liquid during use Consult the enclosed Material Safety Data Sheet for additional details 6 Quick Protocol for Experienced Users Preparation of Competent Cells 1 Inoculate a single colony in 4 ml LB or CIRCLEGROW Broth and grow overnight with shaking at 37 C 2 Prepare a 1 50 dilution of the overnight culture in SUPER COMP Media to provide sufficient volume for 1 ml for each transformation and 5 ml
4. retransform Repeat vector dephosphorylation under conditions that will prevent nibbling the DNA termini Re purify the vector and insert using the GENECLEAN Turbo Kit 1102 200 or via standard phe nol chloroform extraction and precipitation to remove factors that could potentially interfere with ligation Transformation was inefficient or compromised Review issues related to supercoiled plasmid transformation listed in Section 9 1 9 3 have too many colonies or a bacterial lawn on my transformation plate The efficiency of the protocol may have been underestimated Repeat the procedure with less DNA if a lawn is observed Dilute the transformed cells or plate a smaller volume Adjust the formula for calculating efficiency accordingly Determine transformation efficiency from the positive control to confirm kit operation and create a guideline for the expected number of transformants Some of the colonies may be bacterial contamination Any Questions Call Technical Support at 800 424 6101 17 Transform amp Grow Bacterial Tre Restreak the original bacterial stock on selective and nonselective media to confirm purity Prepare fresh competent cells using a pure bacterial culture Resterilize glassware used in the growth and cell preparation Effectiveness of the positive selection may be reduced Analyze the growth media plate containing the positive control bacteria It should not have any colonies If it has colo
5. www gbiogene com
6. 0 preps 2067 200 RapidPURE Plasmid Mini 96 Kit 96 preps 2067 400 RapidPURE Plasmid Mini 96 Kit 4 x 96 preps 2067 600 RapidPURE Plasmid Mini 96 Kit 10 x 96 preps 2069 400 Yeast RPM Kit 100 preps 2000 200 Express Matrix 1 250 preps 2002 200 96well Prep Express 384 preps 2002 400 96well Prep Express 960 preps 2005 200 RapidPURETM Plasmid Midi Kit 25 preps 2005 400 RapidPURETM Plasmid Midi Kit 15 preps 2005 600 RapidPURETM Plasmid Midi Kit 150 preps 2076 200 RapidPURE Plasmid Maxi GF Kit 20 preps 2074 200 RapidPURE Plasmid Maxi GF Endo Free Kit 10 preps 2078 200 RapidPURE Plasmid Giga Kit 12 preps 12 2 Other Transformation Kits and Tools Cat Description Size 2100 200 EZ Yeast Transformation Kit 200 preps 2200 200 Alkali Cation M Yeast Transformation Kit 250 preps 2210 200 Yeast Spheroplast Transformation Kit 25 preps 3103 014 10x RbCl CaCl Transformation Salts 150 ml 5000 552 Roll amp Grow Plating Beads 2 Tubes Any Questions Call Technical Support at 800 424 6101 19 Transform amp Grow Bacterial Tra 12 3 Molecular Biology Certified Growth Media Cat Description Size 3000 112 CIRCLEGROW Powder 227 g 0 5 Ib 3000 165 CIRCLEGROWP Pack 10 x 0 5 L Pouches 3000 121 CIRCLEGROW Capsules 250 Caps 3000 104 CIRCLEGROWP Sterile Solution 500 ml 3002 012 LB Medium Powder 227 g 0 5 Ib 3002 065 LB Medium Pack 10 x 0 5 L Pouches 3002 011 LB Medium Capsul
7. 8 transformants per microgram plasmid DNA but these results are often not reliably reproduced The Qbiogene Transform amp Grow Bacterial Transformation Kit has been developed to minimize the number of reagents procedure steps and hands on time required to reliably obtain consistent transformation efficiency The Transform amp Grow Bacterial Transformation Kit provides a simple and efficient method for the prepa ration transformation and storage of competent E coli cells The transformation technique based on obser vations by Chung et al 5 grows bacterial cells at 37 C harvests them in mid log growth and resuspends them in Transform It Solution This unique buffer both enables the cells to become immediately available for transformation and permits freezing the cells for future use without further preparation The competent 6 visit us on the web at www gbiogene com Bacterial Transformation Kit cells remain stable at 70 C to 80 C for at least 10 weeks The Transform amp Grow Bacterial Transformation Kit eliminates the requirement for heat shock and prepares competent cells within two and a half hours with only 30 minutes of hands on manipulation The kit contains the necessary reagents to pre pare competent cells with a reliable and reproducible transformation efficiency of 2 8 x108 transformants per ug of supercoiled plasmid DNA In addition to the unique Transform It Solution and BIO 101 S
8. Confirm the plates were produced with the correct concentration of antibiotic e g diluted correctly for final concentration Plates were not incubated at the optimal temperature for at least 24 hours 16 visit us on the web at www qbiogene com i Bacterial Transformation Kit Incubating bacteria above approximately 39 C will prevent bacterial growth Check the incubator tem perature with a calibrated thermometer If using a bulb thermometer place it into water in a beaker that has been equilibrated to incubator temperature before reading the value Incubating bacteria below 37 C will slow the growth rate to require longer incubation Determine the incubator temperature as described above If the incubator was below temperature continue the incu bation at the correct temperature and or repeat the transformation 9 2 Why don t I have colonies on my ligation transformation plate Ligation efficiency influenced the transformation outcome Analyze a portion of the ligation reaction by agarose gel electrophoresis to determine if insert has been ligated to vector Increase the amount of ligation transformed to compensate for potential low ligation efficiency Pellet the remaining 900 ul of recovered cells by centrifugation at 5000 x gfor 10 minutes Remove most of the supernatant and resuspend pellet in remaining volume Plate this volume onto the selec tive media Repeat the ligation with increased amounts of vector and or insert and
9. Transform amp Grow Bacterial Transformation Kit For rapid and user friendly preparation of transformation competent bacterial cells Revision 4 2150 999 3C01 Bacterial Transformation Kit Transform amp Grow Bacterial Transformation Kit For rapid and user friendly preparation of transformation competent bacterial cells Application Manual Revision 2150 999 3C01 Catalog 2150 200 50 Samples Storage Ambient temperature 15 30 C Any Questions Call Technical Support at 800 424 6101 3 1 Introduction s ccccncddnadsnddoendcaddiendcndianedsateemnadadaamaend 6 2 Kit Components and User Supplied Materials 7 2 1 Transform amp Grow Bacterial Transformation Kit Components 7 2 2 User Supplied Materials 2 arto triciclo 7 3 Important Considerations Before 8 3 1 Aseptic 8 3 2 Culture Growth Conditions nehmen 8 3 3 en 8 3 4 BOSE ee EE EE ai 8 3 5 Transform It Solution 9 3 6 Incubation TIMO ER 9 3 4 Recommended Controls for All Transformations sees 9 3 8 Recommended Controls for Ligation Transformations 9 4 How to Calculate Transformation Efficiency 10 5 Safety Precautions seen anne 10 6
10. c resistance gene Note Some plasmid host combinations will provide 30 40 of the total possible number of transformants with no incubation step However optimal transformation efficiency is achieved after incubation for 60 minutes 8 Pre warm the appropriate number of selective agar plates at 37 C 9 Transfer 100 ul of the recovered cells onto the pre warmed plates and evenly disperse them using Roll amp Grow Plating Beads as follows A Aseptically 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 disperse the cells Multiple plates may be stacked and rolled simultaneously C Remove the lid and roll beads from the plate into a biohazard waste receptacle 10 Replace the lid and incubate the plates inverted at 37 C overnight 8 Common Questions 8 1 Does Transformation Efficiency Vary with the Volume of Competent Cells Produced The transformation efficiency of the Transform amp Grow Bacterial Transformation Kit does not significantly fluctuate with the total volume of competent cells produced Expected transformation efficiency normally varies between 2 8 x 106 transformants per microgram DNA 8 2 Does Transformation Efficiency Vary with Plasmid Size It is well known that there is an inverse relationship between transformation efficiency and plasmid size For example there can be a tenfold decrease i
11. ells reach a den sity at of 0 5 0 6 Note Harvesting the cells at ODgoo of 0 5 0 6 is recommended for optimal transformation effi ciency Different E coli strains will vary in the time it takes to reach this point 7 2 Preparation of Competent Cells 1 6 No T Transfer the cells to a sterile centrifuge tube Place on ice for 15 minutes Place the bottle of Transform It Solution and the required number of empty sterile microcentrifuge tubes on ice Pellet the cells by centrifugation at 3 000 x g at 4 C for 15 minutes Discard supernatant Determine the volume of Transform It Solution required to resuspend the pellet Each transformation reaction requires a liquid volume of 100 ul of resuspended cells To calculate the volume of Transform It Solution required in milliliters multiply the total number of reactions required determined in Section 7 1 Step 3 by 0 1 For example If 10 transformations are required 10 ml of culture will remain after removing 5 ml to determine the ODgog Section 7 1 Step 3 The cell pellet will therefore be resuspended in 1 ml of Transform It Solution 10 ml x 0 1 ml Resuspend the pellet in the volume of ice cold Transform It Solution determined in Step 4 Incubate the resuspended cells on ice for 15 minutes te If transformation of DNA is to be performed the same day resuspended cells can be left on ice at this step for up to 60 minutes without affecting
12. ependently replicating episomes 2 Following bacterial cell transformation the plasmid is partitioned to daughter cells during cell division Bacterial transformation methods generally fall into the category of either chemical transformation or elec troporation Both methods begin by treating bacterial cells with specific chemicals to prepare them for DNA uptake make them competent and to enhance the plasmid transfer through the bacterial cell wall Electroporation combines this chemical preparation of bacterial cells with the use of electric potential to introduce DNA into the cell Electroporation provides a transformation efficiency up to 109 transformants per microgram plasmid DNA and is typically required for library screening to satisfy the need for increased clone representation Electroporation is not a critical parameter for success when screening a ligation reaction for a clone containing insert DNA or transforming plasmid into a new host strain Many variations of the chemical transformation method have been developed in different laboratories with the intent of optimizing the transformation process The methods may include specialized buffers may incorporate heat shock or incubation on ice during the transformation process or they may require grow ing the bacteria at a reduced temperature to increase transformation efficiency 3 4 Chemical transfor mation methods are reported to provide a transformation efficiency up to 10
13. es 227 g 0 5 10 3002 014 LB Medium Sterile Solution 500 ml 3002 212 LB Agar Medium Powder 227 g 0 5 10 3002 265 LB Agar Medium Pack 10 x 0 5 L Pouches 3002 211 LB Agar Medium Capsules 227 g 0 5 Ib 3002 204 LB Agar Medium Sterile Solid Solution 500 ml 3103 011 SUPER COMP Media Capsules 100 Capsules 3031 012 SOC Medium Powder 227 g 0 5 lb BIO 101 Systems is well known for providing an excellent selection of high quality growth media in a vari ety of formulations We specialize in formulations for yeast and bacterial genetics and offer more than a thousand recipes and variations Each product is subjected to extensive quantitative testing and is Molecular Biology Certified through qualitative molecular biology based tests such as cell density and plasmid yield Choose from a wide variety of convenient packaging formats Pre mixed powders are avail able in capsule form single use pouches or in bulk sizes Liquid media pre poured plates and custom packaging options are also available If you prefer to make your own media BIO 101 Systems can supply you with raw materials such as tryptones and peptones yeast extract a wide variety of sugars and salts antibiotics and other media additives 20 visit us on the web at www gbiogene com Bacterial Transformation Kit 13 Product Use Limitation amp Warranty Unless otherwise indicated this product is for research use only Purchase of Qbiogene Inc products does
14. for the determination Incubate at 37 C to an ODgoo of 0 5 0 6 Transfer cells to a sterile centrifuge tube and place on ice for 15 minutes Centrifuge at 3000 x g at 4 C for 15 minutes Pour off supernatant Resuspend cells in ice cold Transform It Solution 100 ul for each transformation reaction Chill cells on ice for 15 minutes Transfer 100 ul of cells per transformation reaction to prechilled on ice sterile microcentrifuge tubes DADOS 10 visit us on the web at www qbiogene com Bacterial Transformation Kit Transformation of Plasmid DNA Add DNA to each 100 ul aliquot of chilled cells Incubate on ice for 10 minutes Transfer to room temperature for 10 minutes Return to ice for 10 minutes Transfer cells to a sterile aerated 15 ml culture tube containing 0 9 ml SUPER COMP Media Incubate with moderate shaking at 37 C for 60 minutes Pre warm selective agar plates at 37 C Plate 100 ul of culture using Roll amp Grow Plating Beads Incubate plates inverted at 37 C overnight 01 60 IN LE 7 Detailed Protocol 7 1 Culture Preparation 1 Prepare single colonies for inoculation Day 1 Streak the bacterial strain that will be transformed onto a solid growth media to obtain single colonies Use antibiotic selection if required Incubate the bacterial plate inverted overnight at 37 C to obtain single colonies 2 Prepare initial overnight culture Day 2 Using a
15. hout added plasmid should be plated on positive selection media to confirm the selection is functioning and that the bacteria used to prepare competent cells do not contain plasmid prior to use e Positive Control Transform a preexisting plasmid at a known concentration to confirm cell competency check positive selection and permit calculation of transformation efficiency 3 8 Recommended Controls for Ligation Transformations In addition to the controls suggested in Section 3 7 the following controls are strongly recommended for the transformation of ligation reactions e Untreated parent plasmid is used to indicate the transformation efficiency e Prepared e g restriction digested phosphatase treated etc vector without DNA insert in a ligation reaction lacking ligase is used to indicate the potential background from undigested or self ligating parent vector e Insert alone is transformed to indicate if the parent vector containing the insert was completely removed from the insert prior to ligation and transformation Any Questions Call Technical Support at 800 424 6101 9 Transform amp Grow Bacterial 4 How to Calculate Transformation Efficiency Transformation efficiency is calculated as Colony Forming Units CFU per microgram of DNA Each CFU is the result of being transformed with at least one plasmid However if the amount of plasmid DNA is too high a cell may become transformed with multiple plasmids
16. n efficiency from a 3 kb plasmid to a 14 kb plasmid Transformation efficiency for larger plasmids may be increased by adding an increased amount of plasmid DNA See Figure 1 A titration of plasmid DNA concentrations may be needed to find the optimal concen tration for a specific plasmid It has also been observed that lowering the antibiotic concentration in the growth plate may enhance cell survival with larger plasmids 14 visit us on the web at www qbiogene com v Bacterial Transformation Kit Plasmid Name Plasmid Size Concentration Range ng Transformation Efficiency pUC18 2686 bp 5 1000 41 7 1x 108 pBKRSV 4 4 kb 10 50 3 0 5 4 x 108 pGEXAT 4 696 kb 5 10 1 2 1 6x 106 PlasmidMA 6 5 kb 25 1 3 x 108 PlasmidMB 6 7kb 25 1 4 x 106 pCMV Luciferase 9 kb 25 42 6 7 x 108 PlasmidPLRC 12 8 kb 300 1 5 x 106 CosmidMA 45 kb 1000 1500 5 3 7 1 x 10 CosmidMB 45 kb 500 1000 14 17 106 CosmidMC 45 kb 1000 1 8 105 CosmidMD 45 kb 1000 7 7 x 105 Figure 1 Relationship between plasmid size DNA concentration and transformation efficiency 8 3 Do Plasmids Containing Different Antibiotic Resistance Genes Have Different Transformation Efficiencies Transformation efficiency is not dependant upon the selective marker carried by the plasmid used in the transformation procedure Any Questions Call Technical Support at 800 424 6101 15 Transform amp Grow Bacterial Tr 9 Troubleshooting 9 1 Why don t I have colo
17. nies check for contamination See above and repeat the transformation with fresh selec tive medium plates Ensure that the correct concentration of antibiotic was used to prepare the plates and that the plates were used shortly after preparation Ensure the agar was cooled to approximately 55 C warm to the touch prior to adding antibiotic and pouring plates Vector contaminating background is higher than expected Analyze the transformation plate representing the control vector without ligase Colonies present on this plate indicate vector only background that will contribute to background colonies in the vector with insert ligation transformation 9 4 What if the colonies seem too large Larger than expected colonies are not a cause for concern We recommend analyzing the colonies if all con trols fall within expected results Large colonies may be due to incubation at temperatures slightly higher than the recommended 37 C and or incubation of the plates for greater than 18 24 hours Variability in the bacterial plates e g more media less antibiotic or reduced antibiotic activity may also account for larger than normal colony size 9 5 What if there are two or more types of colonies Two types of colonies typically indicate contamination This is especially true if they have different morphol ogy color or size see Section 9 4 While it may be possible to pick a single colony that looks most like the anticipated bacteria and st
18. nies on my supercoiled plasmid transformation plate DNA was not added to the transformation mix Review DNA origin Was plasmid or insert DNA in the original tube Was it possibly mislabeled Was it an old ligation tube that did not produce colonies before Ensure DNA was added to the transformation mix Ensure plasmid DNA was properly diluted to provide 1 10 nanograms per transformation Ensure the proper amount of DNA was added if relying on an ODogo reading for concentration Analyze an aliquot of the DNA by gel electrophoresis to confirm concentration size and integrity DNA was not stored correctly and has degraded Store purified DNA at 20 C to prevent DNase digestion Repurify the DNA from bacteria Analyze an aliquot of the DNA by gel electrophoresis to confirm concentration size and integrity Retransform if the DNA does not appear completely degraded Cells were not made competent Repeat the transformation with a positive control Use a preexisting supercoiled plasmid at a known concentration to confirm cell competency Calculate the transformation efficiency Ensure instructions in the Detailed Protocol Section 7 were followed correctly Incorrect antibiotic selection was used in the plate Review the known antibiotic resistance of the plasmid Confirm growth media plates contain the correct selection Streak plates with a host cell containing a plasmid with the correct selection resistance for confirmation
19. 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 liability 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 CIRCLEGROW and BIO 101 Systems are registered trademarks of Qbiogene Inc Transform amp Grow Transform It Roll amp Grow Alkali Cation and RapidPURE are trademarks of Qbiogene Inc Any Questions Call Technical Support at 800 424 6101 21 Transform Grow Bacterial Transfort NOTES 22 visit us on the web at
20. nsform amp Grow Bacterial Tr 3 Important Considerations Before Use 3 1 Aseptic Technique Practice aseptic technique when using the Transform amp Grow Bacterial Transformation Kit e Minimize the introduction of contamination into the cell culture from dust hair and clothing e Treat the benchtop work area prior to use with 10 bleach or 70 ethanol to remove potential contaminants e Use sterile microcentrifuge tubes glassware plates pipettes and tips e Flame sterilize open liquid containers before and after removing the cap e Aliquot reagents to prevent contaminating the master stock 3 2 Culture Growth Conditions Adequate aeration of the growing bacteria is critical The media volume should be 25 or less of the cul ture container and the inoculated culture should be agitated to provide maximal air transfer to the media Harvesting bacterial cells during middle log phase ODgon of 0 5 0 6 yields optimal transformation efficiency Cells can be harvested during stationary phase but transformation efficiency will be significantly reduced The Transform amp Grow Bacterial Transformation Kit protocol allows for the use of 5 ml of the cell cul ture for determining the bacterial ODggg reading for harvest Cell density may double within 10 15 min utes during the log growth phase 3 3 Plasmid DNA Transformation efficiency is dependent on plasmid size concentration and degree of supercoiling and may need to be tit
21. rated for maximal efficiency In general the transformation efficiency will plateau at approximately 500 nanograms of plasmid DNA As little as 5 nanograms of plasmid will provide approximately 1 4 x 10 colonies with the Transform amp Grow Bacterial Transformation Kit The transformation efficiency of ligated DNA is approximately 100 fold less than covalently closed super coiled DNA When transforming a ligation reaction it is recommended that 1 ul Sul and 10 ul of a 25 pl reaction are used to accommodate variability in the ligation efficiency 3 4 Agar Plates It is strongly recommended that growth plates used for plating the transformation reaction be pre warmed to 37 C before use Cold plates have been experimentally demonstrated to result in reduced transformation efficiency 8 visit us on the web at www qbiogene com Bacterial Transformation Kit 3 5 Transform ItTM Solution The Transform It Solution is stored at room temperature This reagent requires chilling on ice before use but should be returned to room temperature for storage 3 6 Incubation Time The Transform amp Grow Bacterial Transformation Kit permits flexible incubation times Cells resuspended in Transform It M Solution may be incubated on ice for 15 60 minutes prior to the addition of DNA with out significantly affecting the transformation efficiency 3 7 Recommended Controls for All Transformations e Negative Control Competent bacteria wit
22. reak for isolation it is best to repeat the transformation and ensure sterility It is important to note however that the presence of microcolonies that otherwise look like the larger colonies may indicate a phenotype associated with the transformed plasmid Individual cells might respond differently to varying expression levels of a recombinant gene visit us on the web at www gbiogene com i Bacterial Transformation Kit 10 Recommended Reference Format for Publications Bacteria cell name was transformed using the Transform amp Grow Bacterial Transformation Kit Qbiogene Inc CA 11 References Mandel and A Higa 1970 J 53 159 162 Cohen S N Chang A C Y and L Hsu 1972 Proc Natl Acad Sci U S A 69 2110 2114 Hanahan D 1983 J M B 166 557 580 Studies on transformation of Escherichia coli with plasmids Inoue H Nojima H and H Okayama Gene 1990 High efficiency transformation of Escherichia coli with plasmids Chung C T Niemela S L and R H Miller 1989 Proc Natl Acad Sci U S A 86 2172 2175 One step preparation of com petent Escherichia coli Transformation and storage of bacterial cells in the same solution AAR WHS 12 Related Products 12 1 Plasmid Purification Kits Cat Description Size 2066 200 RapidPURETM Plasmid Mini Kit 60 preps 2066 400 RapidPURETM Plasmid Mini Kit 120 preps 2066 600 RapidPURETM Plasmid Mini Kit 30
23. rmation of supercoiled plasmid DNA prepare a dilution such that amounts of 1 ng 5 ng and 10 ng be added using volumes between 5 ul and 20 ul Overloading the cells with plasmid is not recommended and can lead to a decrease in efficiency and or result in a single cell taking up more than one plasmid While a standard pUC based vector may show increased efficiencies for up to 1000 ng transformed the addition of more DNA in order to increase efficiency must be titrated for each plas mid host combination For transformation of ligation reactions volumes of 1 ul 5 ul and 10 pl are recommended In general more DNA is required for efficient transformation of a ligation reaction containing open circular and or nicked DNA than for supercoiled DNA 2 Add plasmid DNA to each 100 ul aliquot of chilled competent cells 3 Gently finger flick tubes to mix and incubate on ice for 10 minutes 4 Transfer tubes to room temperature for 10 minutes 5 Place tubes back on ice and incubate for 10 minutes 6 Add 0 9 ml SUPER COMP Media to a sterile aerated 15 ml culture tube Any Questions Call Technical Support at 800 424 6101 13 Transform amp Grow Bacterial 7 Using aseptic technique transfer the entire volume of transformed cells to the 0 9 ml SUPER COMP Media and incubate at 37 C in an environmental shaker incubator at 100 rpm for 60 minutes Note Incubation permits bacterial cell recovery and expression of the antibioti
24. septic technique transfer 4 ml LB or CIRCLEGROW media to a sterile growth container Inoculate the liquid media with a single bacterial colony from the Day 1 plate and grow overnight at 37 C in an environmental shaker incubator at 200 rpm 3 Grow a sufficient number of cells for transformation Day 3 Each transformation reaction requires a liquid volume of 1 ml of cells at an ODggp of 0 5 0 6 The rec ommended total culture volume required includes an additional 5 ml of culture to permit checking the reading during growth to determine the harvest point See sections 3 7 and 3 8 for recom mended transformation controls As shown in the following table use 1 part overnight culture to 49 parts SUPER COMP Media to pre pare a final culture dilution of 1 in 50 For example if 25 transformation reactions are required a total culture volume of 30 ml is necessary Combine 29 4 ml SUPER COMP Media and 600 ul of the overnight culture This will provide 5 ml of cell culture to check the reading and 1 ml for each of the 25 transformation reactions Any Questions Call Technical Support at 800 424 6101 11 Transform amp Grow Bacterial Tre Volume for Component 5 Reactions 10 Reactions 15 Reactions 25 Reactions SUPER COMP Media 9 8 ml 14 7 ml 19 6 ml 29 4 ml Initial overnight culture for 1 50 dilution 200 ul 300 yl 400 ul 600 ul Incubate the cells at 37 C in an environmental shaking incubator at 200 rpm until the c
25. transformation efficiency If cells are to be frozen for future use do not incubate longer than 15 minutes Proceed with Step 7 and store cells at 70 C While maintaining cells on ice transfer 100 yl per transformation reaction to the chilled sterile micro centrifuge tubes visit us on the web at www qbiogene com Bacterial Transformation Kit 8 Proceed with the transformation protocol Section 7 3 or store the competent cells at 70 C for future use Note Competent cells are stable at 70 C for at least 10 weeks with minimal decrease in trans formation efficiency 7 3 Transformation of Plasmid DNA Note Competent cells prepared immediately prior to use may be stored on ice for up to 60 min utes 45 minutes beyond the recommended 15 minutes before adding DNA without loss of transformation efficiency Section 7 2 Step 6 Note Frozen competent cells should be thawed on ice before proceeding to the next step 1 Prepare DNA for transfer to competent cells Note Refer to Sections 3 7 and 3 8 for appropriate transformation control reactions Note The guidelines for DNA volume given below reflect typical experimental settings for trans formation of ligations and supercoiled plasmids It is only critical to accurately pipet a known quantity of DNA when measurements of transformation efficiency will be determined and compared to other readings or used as standards see Section 3 7 for appropriate controls For transfo
26. ystems SUPER COMP Media the Transform amp Grow Bacterial Transformation Kit includes Roll amp Grow Plating Beads to replace the tra ditional hockey stick and flame method for plating cells on solid growth media The sterile Roll amp Grow Plating Beads are added directly to the growth plate surface with the transformed bacterial cells rolled back and forth to disperse the cells and then discarded prior to incubation 2 Kit Components and User Supplied Materials 2 1 Transform amp Grow Bacterial Transformation Kit Components SUPER COMP Media 120 ml Transform It Solution 6 ml Roll amp Grow Plating Beads 1 each Short Protocol 1 each User Manual 1 each MSDS 1 each Certificate of Analysis 1 each Transform It M Solution is stored at room temperature but should be chilled on at least 15 minutes prior to use 2 2 User Supplied Materials LB Luria Bertani Broth or CIRCLEGROWP liquid culture media see Related Products Section 5 ml sterile culture tubes for growth of the overnight bacterial culture 50 ml or larger sterile culture tubes or flasks for growth of diluted overnight culture 1 5 or 2 0 ml microcentrifuge tubes for the transformation reaction 15 ml sterile aerated polypropylene culture tubes for cell recovery Agar plates for bacterial culture 37 C environmental shaking incubator Pipettmen and tips Ice Spectrophotometer Any Questions Call Technical Support at 800 424 6101 7 Tra
Download Pdf Manuals
Related Search