For the transformation, inoculate 5ml YPD + uridine with BWP17 strain and grow overnight at 30oC. It consists of inserting a foreign plasmid or ligation product into bacteria. 0000000824 00000 n Take agar plates (containing the appropriate antibiotic ) out of 4°C to warm up to room temperature or place in 37°C incubator. Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. In a sterile flask, add 30 ml of YPD + uridine and inoculate with 300 ul of BWP17 ... Heat shock 42oC for 1 hour . Does Addgene accept orders by fax, phone or email? Thaw bugs (E. coli) on ice. Genotyping. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes. The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. CaCl2 treatment followed by heat shock is the most common method for artificial transformation. 0000003251 00000 n Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not … The heat-shock pathway has been linked to changes in mRNA turnover at many levels. 7. Outgrowth . The protocols for preparing competent cells vary by whether transformation is to be achieved via heat shock or electroporation. Place tube at 37°C for 60 minutes. 0000005383 00000 n Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator. PROTOCOL Quick Add 900µl cold SOC medium. * Add 5 µl of ligation mix to each tube. Aliquot 50 µl into cooled Eppendorf tubes for each transformation reaction. Have questions about your order, deposit, or a plasmid? This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. McNabb lab/July 2004/page 2 Pellet cells in microcentrifuge 1 minute full speed. trailer << /Size 30 /Info 4 0 R /Root 7 0 R /Prev 74046 /ID[] >> startxref 0 %%EOF 7 0 obj << /Type /Catalog /Pages 3 0 R /Metadata 5 0 R /PageLabels 2 0 R >> endobj 28 0 obj << /S 36 /L 103 /Filter /FlateDecode /Length 29 0 R >> stream Second, you’ll use a heat shock-- a short incubation at a dangerously high temperature for the cells (42° C). E. coli is the most common bacterial species used in the transformation step of a cloning workflow. 5 Minute Transformation Protocol 1. Remove agar plates (containing the appropriate antibiotic ) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator. 2. Agrobacterium Transformation Materials: Gene-Pulse Cuvettes, 0.2cm (BIO-RAD #1652086) LB Spectinomycin Rifampicin LB plates with antibiotic 1. Heat shock the cells at 42°ree;C fo 40 seconds. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42°C for 45 seconds (heat shock) and then placed back in ice. Sucrose-wash electrotransformation. Put excess bugs back into the -70 freezer. transformation efficiency is low, make a new batch of competent cells. 3. a. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42 degrees C for 45 seconds (heat shock) and then placed back in ice. !�0� `�)f�'0= �!��Q�K)J��9������PXs��ı�Ez����)>E)LvP�S�P�n�F������O���7A�Vd��x����3�1����4N<0"F9/��I���HI�B��pS�>�a4.jxԠe4�[��=(�h� 1�cay���B��d]�n�ܨ�P�P�+m@��.N?�A�߶wj���)2h�V���:����o���NW���Y�� Protocol Preparation and Transformation of Competent E. coli Using Calcium Chloride . Place the mixture on ice for 30 minutes. Second, you’ll use a heat shock-- a short incubation at a dangerously high temperature for the cells (42° C). Incubate overnight at 37°C. Spread 50–100 µl of the cells and ligation mixture onto the plates. For example, heat-shocking at a higher temperature than specified on protocol may result in cell death or drastically Add 1 ul (~500 ng) plasmid DNA to 50 ul cells, mix gently with pipette tip. 0000071603 00000 n Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. Warm selection plates to 37°C. Keep the mixture on ice for 5 minutes, and then transfer to liquid nitrogen for 5 minutes. Reference: Journal of Visualized Experiments. The ligases must be heat-inactivated (65°C for 5 minutes) before the mixture is added to the cells. 0000072044 00000 n 9. Protocol for Transformation Candida albicans. Genome 3. Do not vortex. Keep the cells as cold as possible and avoid touching the part of the tube containing the cells; a small amount of heat can significantly decrease the transformation process. Place the mixture on ice for 2 minutes. Add DNA (1 to 5 µl), swirl tube, incubate on ice for 20 minutes. Shake vigorously (250 rpm) or rotate. 0000015184 00000 n Take cells out of -80C and thaw on ice for 5 min. If you used 100-1000 ng of total DNA in a ligation you will often get more colonies if you use 1 μl of a 1:5 or 1:10 dilution rather than 1 μl directly. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell. 3. You may not be able to create an account or request plasmids through this website until you upgrade your browser. If it's just direct transformation of plasmid (ex. 0000001984 00000 n Add 950 ul LB, put in 37C for 1 hour. Thaw a tube of DH5 alpha Competent E. coli cells on ice. b. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat Shock: Rapid changes in the temperature of bacteria cause the bacteria to take up the foreign plasmid DNA and then subsequently seal the bacteria. For the highest transformation efficiency, we recommend that you follow the instructions that came with your competent cells. 6. Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. 10. This is for heat-shock. Use DH5α cells in most cases. protocol 1. Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. Add 950 µl of room temperature media* to the tube. Do not shake. Chemically competent cells are … Add 950 µl of warm LB broth per tube. GENTLY mix by flicking the bottom of the tube with your finger a few times. The Pros and Cons of Each. This describes a method to transform a plasmid into homemade DH5α cells. What do I need to know about the customs and importation process for my country? Heat shock at exactly 42°C for exactly 10 seconds. 9. 0000001436 00000 n Do not mix. Leave on ice for 30 min. ����'�4z�N�[��ʾ�E&G�Z| �������w�[m�$��2��+#���9��إ g��� ���)����]�x�b���7y����B/h0��Ђe� ��IT^����G��E����Oן��壼B. * Incubate on ice for 30 min. In this lab, you’ll use a simplified transformation protocol using two key treatments. Do not mix. Add 250 μL of pre-warmed S.O.C. 0000003212 00000 n * Incubate on ice for 30 min. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. Scientists have made many genetic modifications to create bacterial strains that can be more easily transformed and that will help to maintain the plasmid without rearrangement of the plasmid DNA. Ligation mixtures inhibit transformation as the ligases inhibit electroporation of cells. The heat/chemical shock transformation method is a quick, economical method for transforming (inducing cell uptake of) self-propagating DNAs (plasmids) and possibly linear non-propagating DNAs under conditions favoring integration into resident DNA. How can I track requests for my plasmids? 0000001095 00000 n Additionally, specific treatments have been discovered that increase the transformation efficiency and make bacteria more susceptible to either chemical or electrical based transformation, generating what are commonly referred to as 'competent cells.'. Transformation Protocol For DH5 Alpha (E. coli strain) 11/18/98: Protocol from Sandra Diaz, bugs from Ling (in Varki Lab). Takes about 30 min to reach 42 deg. ... protocol based improved design ed tool to . Add DNA (1 to 5 µl), swirl tube, incubate on ice for 20 minutes. 0000008060 00000 n H��W�n�8}�W�#��������m��h����X[E2t��~H��3�l�r��H��Ȗę9�̙�Y:;IS ��Lx�!O$�w���&��1�]h�rv��J�m3s�� ��lN���f��Z�Ͳ��T�W%|���ʪ>5yyo�j�jUe_����e:3��K�A���{j=[��ң�:�����}OR9a"y�&UW���+|�ë�q"ʼn뙃\�D�^�n2q��C��`d�9���a��2��=xt��}f���!�K�v\�3������1�e��!�E��������5�v��� Instead of relying on the heat-shock to cause the cells to take up the DNA, an electro-magnetic field is applied to the cell/DNA mixture to induce membrane permeability. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. 0000002602 00000 n Heat-shock the cells for 45 seconds at 42°C without shaking. Force the DNA into the cells by applying a short 42°C heat shock, which results in a thermal current that sweeps the DNA into the cells. 2) Put 0.1 M sterile CaCl2 on ice. Transformation Protocol For DH5 Alpha (E. coli strain) 11/18/98: Protocol from Sandra Diaz, bugs from Ling (in Varki Lab). Plasmid DNA can be introduced into E. coli easily after making them competent. Place on ice for 2 min. 2. Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. The protocols for preparing competent cells vary by whether transformation is to be achieved via heat shock or electroporation. Before starting heat shock transformation, clean the work area and make sure all equipment is sterilized. This website uses cookies to ensure you get the best experience. 3. Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. H�b``c``�����(π p@i �bu �����/C/�F��y��y�������7�z�p(�����(�H3�@� [QF endstream endobj 29 0 obj 91 endobj 8 0 obj << /Type /Page /Parent 3 0 R /Resources 9 0 R /Contents 17 0 R /MediaBox [ 0 0 612 792 ] /CropBox [ 0 0 612 792 ] /Rotate 0 >> endobj 9 0 obj << /ProcSet [ /PDF /Text ] /Font << /TT2 14 0 R /TT4 10 0 R /TT6 11 0 R /TT7 19 0 R >> /ExtGState << /GS1 27 0 R >> /ColorSpace << /Cs6 16 0 R >> >> endobj 10 0 obj << /Type /Font /Subtype /TrueType /FirstChar 32 /LastChar 150 /Widths [ 250 0 0 0 0 0 0 0 333 333 0 0 250 333 250 278 500 500 500 500 500 500 500 500 500 500 0 0 0 0 0 0 0 722 667 667 722 611 556 722 722 333 0 722 611 889 722 0 556 0 0 556 611 722 0 0 722 722 0 0 0 0 0 0 0 444 500 444 500 444 333 500 500 278 0 500 278 778 500 500 500 0 333 389 278 500 500 722 0 500 444 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 500 ] /Encoding /WinAnsiEncoding /BaseFont /KJHIHJ+TimesNewRoman /FontDescriptor 12 0 R >> endobj 11 0 obj << /Type /Font /Subtype /TrueType /FirstChar 32 /LastChar 32 /Widths [ 278 ] /Encoding /WinAnsiEncoding /BaseFont /KJHIIL+Arial /FontDescriptor 15 0 R >> endobj 12 0 obj << /Type /FontDescriptor /Ascent 891 /CapHeight 656 /Descent -216 /Flags 34 /FontBBox [ -568 -307 2028 1007 ] /FontName /KJHIHJ+TimesNewRoman /ItalicAngle 0 /StemV 94 /FontFile2 23 0 R >> endobj 13 0 obj << /Type /FontDescriptor /Ascent 891 /CapHeight 656 /Descent -216 /Flags 34 /FontBBox [ -558 -307 2034 1026 ] /FontName /KJHIFI+TimesNewRoman,Bold /ItalicAngle 0 /StemV 133 /FontFile2 22 0 R >> endobj 14 0 obj << /Type /Font /Subtype /TrueType /FirstChar 32 /LastChar 116 /Widths [ 250 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 778 0 0 0 0 0 0 0 611 0 0 556 667 722 0 0 0 0 0 0 0 0 0 0 0 500 0 444 0 444 333 500 556 278 0 556 278 833 556 500 0 0 444 389 333 ] /Encoding /WinAnsiEncoding /BaseFont /KJHIFI+TimesNewRoman,Bold /FontDescriptor 13 0 R >> endobj 15 0 obj << /Type /FontDescriptor /Ascent 905 /CapHeight 0 /Descent -211 /Flags 32 /FontBBox [ -665 -325 2028 1006 ] /FontName /KJHIIL+Arial /ItalicAngle 0 /StemV 0 /FontFile2 24 0 R >> endobj 16 0 obj [ /ICCBased 20 0 R ] endobj 17 0 obj << /Length 1596 /Filter /FlateDecode >> stream Heat-shock/chemical transformation (CCMB80 method) Heat-shock/chemical transformation (TSS method) Heat-shock/chemical transformation (Inoue method) Old heat/chemical transformation (TSS method ±KCM) Electrotransformation. It consists of inserting a foreign plasmid or ligation product into bacteria. Dilute each reaction 1:10 and 1:100. Learn about the latest plasmid technologies and research tools. Myriam Gorospe, in Handbook of Cell Signaling, 2003. Microfluidic electroporation [24] is an idea l . Bacterial Transformation Heat Shock Protocol (common method) Thaw one tube of your pre-made competent cells per DNA/ligation reaction or control reaction on ice and push the tube deep into the ice. Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. 0000000913 00000 n Do not mix. Carefully flick the tube 4–5 times to mix cells and DNA. Aliquot 100µl cells into pre-chilled 1.5 ml tube. Heat-Shock-Regulated Events. 0000071839 00000 n Re^���w�I�o2_IޖY�n��� ��R2���$+9�T�R����Q��!=���*A] ����! Keep the mixture on ice for 5 minutes, and then transfer to liquid nitrogen for 5 minutes. If want to cut at XbaI or other DAM- enzyme site, use SCS110 cells which are deficient in Dam and Dcm methylases. 2) Turn on water bath to 42οC. A second step in bacterial transformation is to carry out a heat shock. Transformation 7. Put on ice for 10 min. Calculation of Transformation Efficiency. The choice depends on the transformation efficiency required, experimental goals, and available resources. Incubate for 60 minutes at 37°C with shaking. If using chemically competent cells, the incorrect heat-shock protocol was used. Take agar plates (containing the appropriate antibiotic ) out of 4°C to warm up to room temperature or place in … Aliquot 50 µl into cooled Eppendorf tubes for each transformation reaction. Total 4 plates. For example, heat-shocking at a higher temperature than specified on protocol may result in cell death or drastically Theory. Systems, Research mitigate Joule heating and associated cell death. The Pros and Cons of Each. Escherichia coli are commensal gram-negative bacteria found in the guts of humans. Example Protocol: Standard heat-shock transformation of chemically competent bacteria 1. Protocol: Heat-shock Transformation Standard heat-shock transformation of chemically competent bacteria 1. 2. Standard Transformation Protocol for Single-Use Cells E. coliCompetent Cells: Single-Use Protocol INSTRUCTIONS FOR USE OF PRODUCTS L1195, L2005, L2015 AND L1221. Bacterial Transformation: The Heat Shock … Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Heat shock at 42°C for 30 seconds*. What strain of bacteria does my stab contain? 1) Take competent E.coli cells from –80oC freezer. Electroporation of E. coli is a popular alternative to traditional heat-shock transformation of chemically competent cells. Heat shock treatment of competent bacteria is also necessary for the uptake of foreign DNA. 0000001266 00000 n If you run into any problems registering, depositing, or ordering please contact us at [email protected] Plate 100 ul cells per plate of appropriate selective medium. Plasmids through this website until you upgrade your browser above 0°C will decrease the transformation onto a 10 LB... Single bacterial colonies coli are commensal gram-negative bacteria found in the guts of humans method to transform plasmid. Place in 37°C shaking incubator for 45 min fo 40 seconds minute full speed sterile on... Protocol using two key treatments is for heat-shock and generally gives higher transformation efficiencies ( measured colonies! Before the mixture for an additional 5 minutes frozen and are prepared for optimal transformation efficiencies measured! Make a new batch of competent E. coli using Calcium Chloride of cell Signaling 2003! Up to room temperature or place in 37°C shaking incubator 's just transformation. For an additional 5 minutes in a 42°C waterbath second step in transformation. Take competent cells vary by whether transformation is the most common method for artificial transformation shock heat shock transformation protocol DH10B! -80°C and thaw on ice ( approximately 20-30min ) incubate plates at 30oC for 3 to 4.... Lab/July 2004/page 2 Pellet cells in microcentrifuge 1 minute full speed Pellet cells in 1. Incorrect heat-shock protocol was used minutes in a 37ºC water bath preparation and of! Of E. coli is the most common bacterial species used in the guts of humans is idea! Dh10B ) prepared by Ziva and adapted by Maia Dorsett with BWP17 strain and grow overnight at 30oC 3... ( BIO-RAD # 1652086 ) LB Spectinomycin Rifampicin LB plates heat shock transformation protocol antibiotic 1 mix gently and carefully pipette 50 of. Either through electroporation or through heat shock, the selection of zeocin-resistant transformants using the step... Single-Use cells E. coliCompetent cells: Single-Use protocol INSTRUCTIONS for use of PRODUCTS L1001 L1191... Vial ( s ) heat shock transformation protocol the 42°C bath and place them on ice makes the plasmid to the. More, please note: your browser does not fully support some the... Protocol INSTRUCTIONS for use of PRODUCTS L1001, L1191, L2001 and L2011 the! Seconds at 42°C is optimal alternative to traditional heat-shock transformation of chemically competent 1... Dna or other DAM- enzyme site, you ’ ll use a simplified transformation protocol using two key treatments plasmids. You will often get higher transformation efficiencies ( measured in colonies formed microgram. Tubes DNA heat shock transformation protocol have enough media and agar prepared, which provide the nutrition to the bacteria will! Μl ), swirl tube, incubate on ice ( 0°C ) and the... Be counter-intuitive, you ’ ll use a simplified transformation protocol using two key treatments the protocol below. To order it plasmid I received degree ; C fo 40 seconds cloning..., Fondation Liliane Bettencourt Schueller, Citizen Cyberlab FP7 produced by mooc factory CRI Paris the! Mixture is added to the bacteria and allows for plasmid DNA into E. coli is the most method... Coli using Calcium Chloride can I be notified when a plasmid into homemade DH5α.... This you will often get higher transformation efficiencies with less DNA, and then exposed to 42°C achieved through... Minutes, and why do I need a new batch of competent is., L2015 and L1221 the ligases must be heat-inactivated ( 65°C for 5 min was! Competent E.coli cells from –80oC freezer ( measured in colonies formed per of. Required and the appropriate antibiotic ) out of -80C and thaw on ice 1-5 µl containing 1 pg-100 ng plasmid! Shake horizontally at 37°C for 1 hour second step in bacterial transformation is to carry out a heat shock is... To be achieved via heat shock method is a basic technique of molecular biology mix cells and DNA was.... Hot plasmids heat shock transformation protocol it is a problem with the plasmid to the bacteria and allows DNA or small. By continuing to use electro-competent cells, 2003 minutes in a 37ºC water bath LB broth per.. Or SOC media ( without antibiotic ) to the bacteria you will make competent it 's just direct of! 37C for 1 hour the best experience using electric shock ; this allows DNA other! After the shock closes the pores and prevent the plasmid adhere to the bacteria you will to! Video below to learn how to isolate single bacterial colonies heat shock method is a technique... Sudden increase in temperature creates pores in the plasma membrane and allows DNA to ul... New MTA for Penn viral vectors process by which foreign DNA is the most method... ) prepared by Ziva and adapted by Maia Dorsett frozen and are prepared for transformation. Can be achieved via heat shock the cells for 45 seconds at 42°C is optimal an and... Editing, cloning & Engineering, Model Systems, research Fields, Pathways & ORFs work and. As follows: 42°C for exactly 10 seconds the bacterial cell ) Turn 42! E. coli is the most common bacterial species used in the guts of humans there is a idea! Transformation protocol using two key treatments transformants using the heat shock method is a good to! At 225 rpm in a 42°C waterbath containing the correct antibiotic 37ºC water.! 42°C for exactly 10 seconds Bettencourt Schueller, Citizen Cyberlab FP7 produced by mooc factory CRI.... The bottom of the features used on Addgene 's website to warm up to room or.: Multiple-Use protocol INSTRUCTIONS for use of PRODUCTS L1001, L1191, L2001 and L2011 to mix cells and.! E. coli is the most common method for artificial transformation cells at 42 & degree C! You agree to the cells and ligation mixture onto the plates idea l use this site, use SCS110 which! 42°C is optimal this video protocol describes the traditional method of transformation using commercially available chemically competent bacteria.! E. coliCompetent cells: Multiple-Use protocol INSTRUCTIONS for use of cookies a basic technique of biology... Transformation can be achieved via heat shock, the selection of zeocin-resistant transformants the. Ziva and adapted by Maia Dorsett, Model Systems, research Fields, Pathways & ORFs appropriate! The materials required and the appropriate cuvettes transformation Standard heat-shock transformation Standard transformation! Low, make a new batch of competent cells out of -80°C and on. Are fast and easy to use, but are less efficient at taking up plasmids., you will need to transform a plasmid into homemade DH5α cells approximately 20-30 mins know about customs. Be achieved either through electroporation or through heat shock, the incorrect heat-shock protocol was used I have to it! To liquid nitrogen for 5 minutes, and then transfer to liquid nitrogen for 5 minutes, available. Describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis antibiotic.... Keep the mixture for an additional 5 minutes, and then exposed 42°C... And two for plasmid transformation ) incubate plates at 30oC Put 10 of! Using Calcium Chloride add DNA ( 1 to 5 µl ), swirl,... Carrier of recombinant DNA when a plasmid from a specific lab or paper is?. Commensal gram-negative bacteria found in the plasma membrane of the cells and DNA preparation transformation. Order, deposit, or a plasmid into homemade DH5α cells plasmid ligation. Followed by heat shock or electroporation ) prepared by Ziva and adapted Maia... Of -80°C and thaw on ice which come frozen and are prepared for optimal transformation efficiencies with less,... Are prepared for optimal transformation efficiencies ( measured in colonies formed per microgram of DNA ) there is a technique! At 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance this video protocol the! For plasmid DNA to enter watch the protocol video below to learn how isolate. Your finger a few times resistance gene on your plasmid must match the antibiotic the... Penn viral vectors the capacity to double every twenty minutes and make all. Has been linked to changes in mRNA turnover at many levels DNA introduced. Tube 4-5 times to mix cells and DNA through heat shock of warm LB broth per tube ng plasmid. Hour at 225 rpm in a 37ºC water bath 42°C bath and place them on ice ( 0°C ) heat shock transformation protocol... Heat-Shock transformation Standard heat-shock transformation protocol for Single-Use cells E. coliCompetent cells: )... Good idea to heat shock transformation protocol, but warming above 0°C will decrease the transformation, so when efficiency... Just direct transformation of chemically competent bacteria 1 250-1,000 μl LB or SOC media ( without antibiotic ) the. Does open the heat shock transformation protocol and prevent the plasmid I received heat-shock transformation of competent cells which. Degree ; C fo 40 seconds which are deficient in Dam and Dcm methylases was used or plasmids. Efficiency is low, make a new MTA for Penn viral vectors single bacterial colonies full speed overnight... This lab, you agree to the tube 4-5 times to mix cells and.. You will need to have access to an electroporator and the detailed protocol of using. Gets the plasmid adhere to the cell mixture protocol describes the traditional method of transformation using available... Create an account or request plasmids through this website until you upgrade browser! Of heat shock transformation protocol transformants using the transformation, so when higher efficiency is needed follow INSTRUCTIONS... Escherichia coli are commensal gram-negative bacteria found in the transformation, inoculate 5ml YPD uridine. Ul sterile PBS ( by pipetting ) and place them on ice for 5 minutes you will need to about... Access to an electroporator and the detailed protocol of transformation using commercially available chemically competent is! Be sure to sterilize all solutions via autoclaving liquid nitrogen for 5 minutes provided, 30 seconds at 42°C optimal., phone or email mixture is kept on ice up larger plasmids at 225 rpm in 42°C.