T4 DNA ligase. Manual for catalog numbers C0005 and C0006. Upon Receipt Store Kits at -20ºC. anvaxbiotech.com

T4 DNA ligase Manual for catalog numbers C0005 and C0006 Upon Receipt Store Kits at -20ºC www.c anvaxbiotech.com PRODUCT MANUAL Version 2.0 Last updated: February 2010

Table of contents Table of contents... ii Materials provided and storage conditions... iii 1-.INTRODUCTION: Main properties of CanvaxT4 DNA ligase... 1 1.1 Description of Canvax T4 DNA ligase.... 1 1.2 Applications of Canvax T4 DNA ligase... 1 1.3 Source, Molecular Weight and Properties of Canvax T4 DNA ligase... 1 1.4 Definition of Activity Units of Canvax T4 DNA ligase.... 1 1.5 Additional Materials Required... 1 2-. PROTOCOLS: Suggested Detailed Protocols for T4 DNA Ligase... 2 2.1 Cohesive Ends Ligation Protocol.... 2 2.2 Blunt Ends Ligation Protocol... 3 2.3 Self Ligation of Linear DNA Protocol...... 4 3-. QUALITY CONTROL... 6 3.1 Quality control assay for Canvax T4 DNA Ligase... 6 3.2 Performance of Canvax T4 DNA Ligase versus other ligases... 6 4-. TROUBLESHOOTING.... 7 5-. FAQs about Canvax T4 DNA Ligase... 8 Licensing and trademark information... iv Notes.... v ii

MATERIALS PROVIDED, KIT STORAGE AND EXPIRATION DATE MATERIALS PROVIDED FOR CAT. NO. C0005 Item Composition Quantity Storage Canvax T4 DNA Ligase* 5 Weiss Units/µL 300U (60 µl) - 20ºC 5x T4 DNA Ligase Buffer 5x 250 µl - 20ºC *Storage Buffer for Canvax T4 DNA Ligase: 20 mm Tris-HCl (ph 7. 5), 50 mm KCl, 1 mm DTT, 0.1 mm EDTA and 50% glycerol. MATERIALS PROVIDED FOR CAT. NO. C0006 Item Composition Quantity Storage Canvax T4 DNA Ligase* 5 Weiss Units/µL 1000U (200 µl) - 20ºC 5x T4 DNA Ligase Buffer 5x 2x250 µl - 20ºC *Storage Buffer for Canvax T4 DNA Ligase: 20 mm Tris-HCl (ph 7. 5), 50 mm KCl, 1 mm DTT, 0.1 mm EDTA and 50% glycerol. Storage temperature: -20ºC in a NON Frost-Free Freezer. Expiration date: See on the kit label. IMPORTANT: T4 DNA ligase is an extremely temperature sensitive enzyme and must be stored at -20ºC in a non-frost free freezer since temperature rises above 0ºC daily in frost-free freezers. If properly stored, kits are guaranteed for at least 9 months from the date of purchase. iii

1. INTRODUCTION: Main properties of Canvax T4 DNA Ligase. 1.1. Description of Canvax T4 DNA Ligase T4 DNA ligase catalyses the formation of a phosphodiester bond between juxtaposed 5 -phosphate and 3 -hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids but has no activity on single stranded nucleics acids. The enzyme requires ATP as a cofactor. 1.2. Applications of Canvax T4 DNA Ligase: Cloning of restriction enzyme generated DNA fragments. Cloning of PCR products. Joining of double-stranded oligonucleotides linkers or adaptors to DNA. Site-direct mutagenesis. Amplified fragment length polymorphism. Ligase-mediated RNA detection. Nick repair in duplex DNA, RNA or DNA/RNA hybrids. Self-circularization of linear DNA. 1.3. Source, Molecular Weight and Properties of Canvax T4 DNA Ligase Source: E. Coli cells carrying a cloned gene 30 from bacteriophage T4. Molecular Weight: 55.3 KDa monomer. Enzyme properties: Reaction temperature from 16ºC at 25ºC (optimal at 22ºC). Heat Inactivation 65º C for 10 min. or at 70º C for 5 min. Chemical Inactivation by NaCl or KCl at concentrations higher than 200 mm. 1.4 Definition of Activity Units of Canvax T4 DNA Ligase. One Weiss unit of the enzyme catalyses the conversion of 1 nmol of [ 32 PPi] into Noritadsorbable form in 20 min at 37º C. One Weiss unit is equivalent to approximately 200 cohesive end ligation (CEL) units and one CEL unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of 1µg lambda DNA in 30 min. at 16º C. Enzyme activity is assayed in the following mixture: 66mM Tris-HCl (ph 7.6), 6.6 mm MgCl2, 0.066 mm ATP, 10 mm DTT, 3.3 µm [ 32 PPi]. 1.5 Additional Materials Required. DNAse, RNAse and protease-free sterile tips, tubes and Pipettes. A water bath at 22ºC (for optimal ligation) and a water bath at 65ºC to 70ºC for heat inactivation of Canvax T4 DNA Ligase after ligation. An ice bath for storage of enzyme during use. A vortex for buffer resuspension. A microcentrifuge for brief spin of enzyme before use. 1

2-. PROTOCOLS: Suggested Detailed Protocols for T4 DNA Ligase ADVICE: Before use of Canvax T4 DNA Ligase it is strongly advised to do the following: 1-. To thaw 5x T4 DNA Ligase at room temperature and vortex it vigorously to mix the components. 2-. To make aliquots of both Canvax T4 DNA Ligase and 5x T4 DNA Ligase Buffer to avoid contamination with nucleases. 3-. To spin the vial of Canvax T4 DNA Ligase for a few seconds before pipetting the enzyme. EXTREMELY IMPORTANT: 5x T4 DNA Ligase Buffer contains ATP, which degrades during temperature fluctuations. It is frequent to see a white precipitate on 5x T4 DNA Ligase Buffer. Please do not try to heat to dissolve the precipitate of 5x T4 DNA Ligase buffer as the ATP will be degraded. 2.1 Cohesive Ends Ligation Protocol for Canvax T4 DNA Ligase. 1-.Prepare the following mixture: 10-20 ng precut DNA Vector with non-compatible cohesive ends x ng precut insert (molar ratio of vector to insert is usually from 1:1 to 1:5. See below). 2 µl 5X T4 DNA Ligase Buffer 1 µl T4 DNA Ligase (5 U/ µl) up to 10 µl water nuclease-free 2-.Incubate for 30-60 min. at 22º C. (The overall number of colonies may be increased when reaction time is prolonged to 60 minutes and after that no additional benefit is reached). 3-. Heat inactivate at 70ºC for 5 min. (Optional) 4-. Use 5 to 10 μl of the mixture for transformation of 50 μl of chemically competent cells. ADVICE: It is advised to follow the next guidelines: 1-. Do not exceed the recommended amount of T4 DNA Ligase in the reaction mixture. 2-. For efficient transformation, the volume of the ligation reaction mixture should not exceed 10-20% of the competent cell volume. 3-. DNA resolved on agarose gel is generally visualized by illumination with ultraviolet light. Exposure to short wavelength light (e.g., 254, 302, or 312 nm) for 2 minutes reduces the ligation efficiency of DNA up to 10.000 times due to formation of pyrimidine dimmers. We strongly recommended the use of a long wavelength lamp (e.g. 360 nm) and the shortest exposure times when isolating DNA from agarose gels for cloning. 2

Example of calculation of amount of insert Vector to insert molar ratios for a succesful ligation may be selected from less than 1:1 to more than 1:5 but the above ratios are the most commonly used. If you choose molar ratio of vector to insert of 1:5, use the following equation: ng insert = pb insert x ng vector pb vector x molar ratio (vector to insert) then pb insert x ng vector ng insert = x 5 pb vector EXTREMELY IMPORTANT: If the ligation reaction mixture will be used for electroporation, a DNA purification step is needed before electroporation to prevent arcing of E. coli cells due to salts contained in the ligation buffer. In this case replace the heat inactivation step with spin column purification or chloroform extraction. 2.2 Blunt Ends Ligation Protocol for Canvax T4 DNA Ligase. Before use of Canvax T4 DNA Ligase see advice on page 2. 1-.Prepare the following mixture: 10-20 ng of DNA Vector whose blunt ends could not be ligated (eg. pspark vectors) x ng of blunt insert (molar ratio of vector to insert is usually from 1:1 to 1:5. See below). 2 µl 5X T4 DNA Ligase Buffer 1 µl T4 DNA Ligase (5 U/ µl) (OPTIONAL )1 µl 10X PEG 6000 (reagent not supplied) up to 10 µl water nuclease-free 2-.Incubate for 30-60 min. at 22º C. (The overall number of colonies may be increased when reaction time is prolonged to 60 minutes and after that no additional benefit is reached). 3-. Heat inactivate at 70ºC for 5 min. (Optional step that can be made only if no PEG 6000 is included in ligation) EXTREMELY IMPORTANT: Do not heat inactivate ligation with PEG because it reduces transformation efficiency. Alternatively you may replace the heat inactivation by chemical inactivation with NaCl or KCl at concentrations higher than 200mM. It is possible to use the mix ligation without inactivation. 3

4-. Use 5 to 10 μl of the mixture for transformation of 50 μl of chemically competent cells. ADVICE: It is advised to follow the next guidelines: 1-. Do not exceed the recommended amount of T4 DNA Ligase in the reaction mixture. 2-. For efficient transformation, the volume of the ligation reaction mixture should not exceed 10-20% of the competent cell volume. 3-. DNA resolved on agarose gel is generally visualized by illumination with ultraviolet light. Exposure to short wavelength light (e.g., 254, 302, or 312 nm) for 2 minutes reduces the ligation efficiency of DNA up to 10.000 times due to formation of pyrimidine dimmers. We strongly recommended the use of a long wavelength lamp (e.g. 360 nm) and the shortest exposure times when isolating DNA from agarose gels for cloning. 4-. Polyethylene glycol (PEG) greatly increases the ligation efficiency of blunt-end DNA ligation. Example of calculation of amount of insert Vector to insert molar ratios for a succesful ligation may be selected from less than 1:1 to more than 1:5 but the above ratios are the most commonly used. If you choose molar ratio of vector to insert of 1:5, use the following equation: ng insert = pb insert x ng vector pb vector x molar ratio (vector to insert) then pb insert x ng vector ng insert = x 5 pb vector EXTREMELY IMPORTANT: If the ligation reaction mixture will be used for electroporation, a DNA purification step is needed before electroporation to prevent arcing of E. coli cells due to salts contained in the ligation buffer. In this case replace the heat inactivation step with spin column purification or chloroform extraction. 2.3 Self Ligation of Linear DNA Protocol for Canvax T4 DNA Ligase. Before use of Canvax T4 DNA Ligase see advice on page 2. 1-.Prepare the following mixture: 1 ng of DNA Vector whose ends could be ligated each other 4

2 µl 5X T4 DNA Ligase Buffer 1 µl T4 DNA Ligase (5 U/ µl) up to 10 µl water nuclease-free 2-.Incubate for 30-60 min. at 22º C. (The overall number of colonies may be increased when reaction time is prolonged to 60 minutes and after that no additional benefit is reached). 3-. Heat inactivate at 70ºC for 5 min. (Optional) 4-. Use 5 to 10 μl of the mixture for transformation of 50 μl of chemically competent cells. ADVICE: It is advised to follow the next guidelines: 1-. Do not exceed the recommended amount of T4 DNA Ligase in the reaction mixture. 2-. For efficient transformation, the volume of the ligation reaction mixture should not exceed 10-20% of the competent cell volume. 3-. DNA resolved on agarose gel is generally visualized by illumination with ultraviolet light. Exposure to short wavelength light (e.g., 254, 302, or 312 nm) for 2 minutes reduces the ligation efficiency of DNA up to 10.000 times due to formation of pyrimidine dimmers. We strongly recommended the use of a long wavelength lamp (e.g. 360 nm) and the shortest exposure times when isolating DNA from agarose gels for cloning. EXTREMELY IMPORTANT: If the ligation reaction mixture will be used for electroporation, a DNA purification step is needed before electroporation to prevent arcing of E. coli cells due to salts contained in the ligation buffer. In this case replace the heat inactivation step with spin column purification or chloroform extraction. 5

3. QUALITY CONTROL: Assays made to Canvax T4 DNA Ligase. Canvax T4 DNA Ligase has passed the following quality control assays: Functional absence of endonuclease and exonuclease activities. exonuclease of T4 DNA Ligases and the white colonies. Ribonuclease assay. Blue/white cloning assay. Effect of the activity endo/exonuclease with other ligase suppliers In this experiment comparing the possible contamination with nucleases of T4 DNA Ligase from Canvax to other ligases of the market. We mix in the ligation reaction 4 ng of pspark I DNA Cloning Vector with a 7 ng of DNA insert with 1 Kb amplified with a proofreading polymerase. We incubate the reaction about 60 minutes at 22º C. We usually obtain about 3500 white colonies and about 10 blue colonies. The amount of blue colonies means an increase in nuclease activity. Because loss of bases in single strand so we can see high number of blue colonies when we use T4 DNA ligases with high nuclease contamination. See the table: Ligase supplier White colonies Blue colonies Canvax 3590 11 Supplier N 890 6 Supplier R 1110 350 Supplier P 1430 110 Supplier I 3980 1160 So we can say that T4 DNA Ligase from Canvax is the best ligase in the market. Because it is highly efficient and it has not nuclease activity. 6

3-. TROUBLESHOOTING. For questions not addressed here, please contact us at www.c anvaxbiotech.com or alternatively contact your local Distributor. PROBLEM CAUSE SOLUTION No colonies obtained 1-The insert and DNA vector have incompatible ends. Confirm that the vector and insert have been digested with the same restriction endonuclease or with restriction endonuclease that generate compatible ends. 2-Low transformation efficiency of E. coli strain. 3-Molar ratio of insert to vector is incorrect. Check transformation efficiency with uncut DNA vector. You must use competent cells with a transformation efficiency of 10 7 cfu/µg DNA to obtain about 500 colonies using 50 pg of DNA vector in the ligation mixture. Check the concentration of DNA by gel electrophoresis and use the recommended molar ratio. (See example of calculation of amount of insert). 4-ATP in the reaction buffer has been degraded. Use a 5X T4 DNA Ligase buffer that is less than one year old and make aliquots of this buffer to avoid ATP degradation. Few or no colonies obtained 5- T4 DNA Ligase has been inactivated. Use a fresh T4 DNA Ligase aliquot. 6-There is an inhibitor of T4 DNA Ligase in the reaction mixture. Be sure that the DNA is free of phenol, that phosphate concentration is <25 mm and that the NaCl concentration is 50 mm. 7-DNA is degraded by contamination with nonspecific endonucleases. Use a fresh buffer aliquot, fresh T4 DNA Ligase aliquot, and autoclaved distilled water. 8-Restriction nucleases are present in the reaction mixture causing redigestion of ligated products Remove restriction endonucleases by phenol extraction or clean-up protocol. 7

Frequently Asked Questions (FAQs) about pspark DNA cloning systems. As a top quality service to our customers, FAQs are continuously updated in our web site (www.c anvaxbiotech.com) so please check the online manual version to see if FAQs have been updated. Q1-. Which are the most common problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure? A1: The volume of ligated DNA was higher than 20% of the competent cell volume. The ligated DNA was degradaded due to a contaminating nuclease in the water or other reagents used in the ligation before transformation. Ligation failed because there was no ATP or Mg +2. Use the supplied buffer. The ATP in buffer older than one year or buffer with multiple freeze-thaw cicles may have degraded enough to cause problems. So, we recommend to make aliquots of the buffer. The ligation failed due to high salt or EDTA in the reaction. Clean up the DNA. The phosphatase used for the dephohorilation step was not completely inactivated.. Follow the recommended procedure to remove the phosphatase. The ligated end was a single base overhangor a blunt end. Use PEG 6000 in the reaction mixture at 5% (w/v). The insert and the plasmid do not have phosphates.check it and if needed order phophorylated primers or alternatively use pspark DNA Cloning Systems. The ligase was inactive. Test on lambda HindIII or other convenient substrate. Q2-.I did not get colonies after transformation protocol, how I can know where is the problem? A2: You should be use the following controls reactions: The cells are not viable. Test the transformation efficiency of the competent cells using a supercoiled vector. Perform a transformation reaction and plate the number of cells that is expected to generate 50-100 colonies per plate, based on the anticipated transformation efficiency of the competent cells. The expected number of colonies should be seen, indicating that the competent cells were transforming with high efficiency. If the expected number of colonies is not seen, please check your competent cells and/or transformation protocol.. Restriction endonuclease-digested, religated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Religation of vectors with the some cohesive ends should result in less than or equal to 50% of the number of colonies obtained with supercoiled vector DNA, indicating that the components of the ligation reaction are working; religation of vectors with blunt ends should yield 10% to 20% of the number of colonies obtained with supercoiled vector DNA. This is an appropriate control only with vectors that have been digested with a single restriction endonuclease; double-digested vectors may not religate because the ends are incompatible and the small DNA fragment that is released from between the two sites is sometimes lost during ethanol precipitation of the DNA. Q3-. I got a high number of background colonies, how I can know where is the problem? A3: You should use the following control reactions: Restriction endonuclease-digested vector. Perform a transformation with an amount of restriction enzymedigested vector DNA equivalent to that contained in the fraction of the ligation reaction being used for the experimental transformations. Few or no colonies should be seen, indicating complete restriction endonuclease digestion of the vector. The presence of colonies indicates incomplete digestion of the vector that will cause a background of colonies containing non-recombinant vector in the experimental transformations. Restriction endonuclease-digested, dephosphorylated, religated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Few or no colonies should be observed, indicating complete dephosphorylation of the vector. A dephosphorylated vector should not be re-ligated by T4 DNA ligase. No DNA transformation control. Perform a mock transformation of competent cells, to which no DNA is added. No colonies should be seen, indicating that the selection antibiotic on the agar plates is potent and that the competent cells are pure. 8

Q4-. Why is ATP present in the reaction buffer for T4 DNA Ligase? A4: ATP is necessary for enzymatic function. It is involved in phosphorylating the ligase prior to the ligation reaction. Ligation efficiency is markedly reduced by removing ATP from the reaction. It is important, therefore, to handle the buffer appropriately in order to minimize degradation of ATP. Q5-. Do both my insert and vector need to be phosphorylated for ligation? A5: At least one molecule in the ligation reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (not dephosphorylated) is routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form. Q6-. Which T4 DNA ligase protocol should be used when ligating an insert containing one cohesive (sticky) end and one blunt end? A6: For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends. The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. Therefore you should use the more stringent protocol for blunt end ligation. Q7-. How can I determine if my ligase is still active? A7: Ligation reagents may be tested by performing a ligation reaction with a molecular size marker such as the 1 Kb DNA Ladder or lambda DNA/Hind III Fragments. Compare the ligation reaction products to unligated DNA on an agarose gel. The ligation reaction should contain a high molecular weight smear and few low-molecular weight bands. If the marker ligation does not work, use a fresh ligase. Another reason for low activity could be degradation of the ATP in the reaction buffer; use 5X ligase buffer that is less than 24 months old. The buffer should be stored at -20 C. Q8-. What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation fail? A8: The restriction enzyme did not cleave efficiently. If cleaving near the end of a PCR fragment leave at least 6 bases past the restriction site. Test the restriction enzyme on a control substrate. The restriction enzyme was not completely inactivated. Phenol/EtOH purify the DNA or Clean-up if the enzyme cannot be heat inactivated. Star activity from the restriction digest cleaved the vector or insert. Check the DNA on a gel. If there is an extraband, reduce the amount of enzyme or time for the restriction digest. The DNA or restriction enzyme contained exonuclease or phosphatase that damaged the ends. Q9-. Why should I take care when electroporating with the ligation reaction? A9: You must purifly the ligaton mix prior to electroporation. The buffer and the T4 DNA Ligase must be removed to prevent arcing of E.coli cells. Try phenol extraction o DNA clean up columns for remove this buffer and T4 DNA Ligase. You must use about 10 μg trna or glycogen carrier for DNA transformation. The electrporator should be optimized for the volume of the mix DNA and competent cells. 9

Warranty: Canvax Biotech SL warrants that the product will meet the specifications stated on this Product Manual and agrees to replace the product free of charge if the product does not conform to the specifications but notice for replacement must be given within 30 days. In consideration of the above promises by Canvax Biotech SL, the purchaser agrees to and accepts the following conditions: (1) That this warranty is in lieu of all other warranties, expressed or implied; (2) That ALL WARRANTIES OF MERCHANTABILITY OR OF FITNESS FOR A PARTICULAR PURPOSE ARE HEREBY EXCLUDED OR WAIVED; (3) That the purchaser's sole remedy shall be to obtain replacement of the product free of charge from Canvax Biotech SL; and (4) That this remedy is in lieu of all other remedies or claims for damages, consequential or otherwise, which the buyer may have against Canvax Biotech SL. Guidelines for Safe Use of the Products: Canvax Biotech SL recommends that the purchaser or user of this product follow the Local Guidelines for Research involving Recombinant DNA Molecules. Canvax Biotech SL disclaims any and all responsibility for any injury or damage which may be caused by the failure of the purchaser or user to follow said local guidelines. See Material Safety Data Sheet (MSDS) on the web site www.canvaxbiotech.com Notice to Purchaser: Information presented herein is accurate and reliable to the best of our knowledge and belief, but is not guaranteed to be so. Nothing herein is to be construed as recommending any practice or any product in violation of any patent or in violation of any law or regulation. It is the user's responsibility to determine for himself or herself the suitability of any material and/or procedure for a specific purpose and to adopt such safety precautions as may be necessary. Prices are subject to change without notice. Product claims are subject to change. Please access the Canvax Biotech SL web site ( www.canvaxbiotech.com for the most up-to-date version of this Product Manual. Important licensing information: Canvax Biotech SL has the exclusive license of patents applications related to pspark DNA cloning systems. The purchase of products based on this technology conveys to the purchaser the non-transferable right to use the purchased product in research conducted by the purchaser (whether the purchaser is an academic or for-profit entity). The purchaser cannot transfer or sell (i) this product or its components or (ii) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The purchaser may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing to use such transferred materials or information only for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. In particular, the use of pspark DNA cloning systems for gene cloning to be used in antigens discovery is expressly prohibited. By use of this product the purchaser accepts the terms and conditions of this limited use statement. If the purchaser is not willing to accept the limitations of this limited use statement, Canvax Biotech SL is willing to accept return of the product with a full refund. For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Canvax Biotech SL at (www.c anvaxbiotech.com) or by e-mail: info@c anvaxbiotech.com Trademark information: Registered names and trademarks used in this document, even when not specifically marked as such, are not to be considered unprotected by law. pspark is a registered trademark of Canvax Biotech SL; GelGreen is a trademark of Biotium Inc.; Dark Reader is a registered trademark of Clare Chemical Research Inc.; FlashGel is a registered trademark of Lonza Group Ltd; NanoDrop is a trademark of Thermo Fisher Scientific Inc. This product is sold for Research or Laboratory Use Only and is not to be used for diagnostic, on humans or for any drug purposes. iv

NOTES. v