Cat. no. EM21 Version: 1.2016 Kit for plasmid DNA isolation in 96-well format EXTRACTME is a registered trademark of BLIRT S.A.
www.dnagdansk.com
Cat. no. EM21 I. INTENDED USE The EXTRACTME PLASMID DNA 96-WELL KIT is designed for high-throughput and efficient purification of high quality plasmid DNA from recombinant Escherichia coli strains. The isolation protocol and buffer formulations were optimized for high isolation efficiency and DNA purity. Additionally, this protocol consists of centrifugation or vacuum based procedure that omits conventional cell pelleting and resuspention steps. The product is intended for research use only. II. COMPONENTS OF THE KIT AND STORAGE CONDITIONS NUMBER OF ISOLATIONS 2x 96 WELL PLATES / KIT 10x 96 WELL PLATES / KIT Catalogue number EM21-192 EM21-960 7x Lysis Buffer 19.2 ml 96 ml Neutralization Buffer 67.5 ml 337.5 ml Wash Buffer (conc.)* 50 ml 5 x 50 ml Elution Buffer 38.5 ml 5 x 38.5 ml Collection Plates 2 pcs 10 pcs Plasmid Binding Plates 2 pcs 10 pcs Plasmid Elution Plates 2 pcs 10 pcs Elution Adhesive Seals 2 pcs 10 pcs * Before using for the first time, add the aropriate quantit y of 96-100% ethanol to the Wash Buffer; for details, see the instructions on the bottle label and in the table below. Mark the bottle after adding the alcohol is recommended. Neutralization Buffer should be stored at +4 C. All the other components of the kit should be stored at room temperature (15-20 C). In order to avoid evaporation, ensure that the buffer bottles are tightly closed before storing. Under proper storage conditions, the kit will remain stable for at least 12 months. 3
NUMBER OF ISOLATIONS 2x 96 WELL PLATES / KIT 10x 96 WELL PLATES / KIT Catalogue number EM21-192 EM21-960 Wash Buffer 50 ml 5 x 50 ml 96-100% ethanol 200 ml 5 x 200 ml Total volume 250 ml 5 x 250 ml III. ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED 96-100% ethanol PFA multichannel pipettes and pipette tips reagent reservoirs for multichannel pipets disposable gloves microcentrifuge with rotor for plates ( 3k x g) dry block heater or water bath (up to 70 C) vortex mixer 96 deep-well plates for bacterial culture and samples preparation adhesive seals vacuum manifold and vacuum pump (producing pressure of -400 to -600 mbar) or automated liquid handling workstations IV. PRINCIPLE The DNA purification procedure utilizes spin 96-minicolumns plates with membranes which efficiently and selectively bind nucleic acids. In the first isolation step, the plasmid DNA is released from bacterial cells by alkaline lysis. Then the lysate is neutralized and all the cell residues along with the proteins and genomic DNA are separated. The lysate is alied to the purification minicolumn membrane and the DNA is bound. The two-step washing stage effectively removes impurities and enzyme inhibitors. The purified plasmid DNA is eluted using a low ionic strength buffer (Elution Buffer) or water (ph 7.0-9.0) and may be used directly in all downstream alications such as PCR, qpcr, Southern blotting, DNA sequencing, enzymatic restriction, ligation and so forth, or stored until ready to use. www.dnagdansk.com 4
Cat. no. EM21 V. QUALITY CONTROL The quality of each production batch (LOT) of the EXTRACTME PLASMID DNA 96-WELL KIT is tested using standard QC procedures. The purified DNA concentration, quality and stability are evaluated by gel electrophoresis and spectrophotometer. In addition, the functional quality is tested by qpcr and digestion with restriction enzymes. VI. PRODUCT SPECIFICATIONS SAMPLE MATERIAL Bacterial broth culture BINDING CAPACITY Arox. 20 µg DNA per well TIME REQUIRED Arox. 35 minutes for purification using centrifuge or vacuum manifold DNA PURITY A 260 /A 280 ratio = 1.7 1.9 5
VII. SAFETY PRECAUTIONS Bacterial culture is treated as a biohazardous material on account of its potential pathogen content or health and life-threatening substances. While working with bacterial cultures, all the safety requirements for working with biohazardous material is essential. Conducting the entire isolation procedure in a Class II Biological Safety Cabinet or at a laboratory burner is recommended, as is wearing disposable gloves and a suitable lab coat. The use of sterile pipette filter tips is recommended. Avoid the cross-transferral of DNA between minicolumns. Guanidine salts residues may form highly reactive compounds when combined with oxidation compounds. In case of spillage, clean the surface with a detergent-water solution. In case of spillage of a liquid containing microorganisms, clean the contaminated surface with a detergent-water solution. VIII. RECOMMENDATIONS AND IMPORTANT NOTES Vacuum manifold use Establish a reliable vacuum source for the EXTRACTME PLASMID DNA 96-WELL KIT vacuum manifold protocol. The manifold may be used with a vacuum pump or water aspirator. Use a vacuum pressure of -400 to -600 mbar or reduce the vacuum pressure until a flow rate 1-2 drops per second is achieved. Using higher vacuum pressure than recommended may cause sample splattering, while using lower vacuum pressure will affect the elution resulting in lower recovery. Keep all unused well in the Plasmid Binding Plate sealed with an adhesive seal during purification to obtain a uniform vacuum and avoid contaminating unused wells. To check the vacuum pressure place an unused Plasmid Binding Plate on top of the vacuum manifold. Seal the plate with an adhesive seal. Aly vacuum and check the vacuum pressure on the vacuum regulator. Adjust the vacuum pressure on the regulator to obtain the recommended pressure. www.dnagdansk.com 6
Cat. no. EM21 Pressure conversions Recommended pressure (mbar) -400 to -600 Conversion from millibars Multiply by Millimeters of Mercury (mmhg) 0.75 Kilopascals (kpa) 0.1 Inches of Mercury (inchhg) 0.0295 Torrs (Torr) 0.75 Atmospheres (atmos) 0.000987 Pounds per Square Inch (psi) 0.0145 Sample material The DNA isolation efficiency and purity can vary depending on a number of factors, such as plasmid copy number (high copy and low copy plasmids), cell density of bacterial culture, cell type (morphology), culture medium, growth phase, age and condition of cells. It is recommended to extract DNA from fresh, properly prepared starting material, which guarantees best isolation parameters. To minimize DNA degradation, avoid subjecting the sample material to repeated freeze/thaw cycles. Using old or repeatedly frozen/thawed material may result in low efficiency of the isolation and poor DNA quality. DNA elution The optimal volume of the elution buffer used should be chosen in line with t h e q u a n t i t y of t h e s a m p l e m a t e r ia l a n d t h e fi n a l D N A co n ce n t r a t i o n e x p e c t e d. The use of 50-100 µl Elution Buffer is recommended. If a high DNA concentration is desired, the elution volume may be reduced to 20 µl. It should be noted that this may reduce the efficiency. It is essential to aly the elution buffer precisely to the centre of the membrane. In order to maximize the DNA retrieval, heat the Elution Buffer to 80 C and incubate it on the membrane for 10 minutes. 7
If full DNA retrieval is required, 200 µl or more Elution Buffer should be used. However it will result in the DNA dilution. Second elution can also be performed. For the second elution, repeat steps 8-11 of the Isolation Protocol while purification using centrifuge (see section XI.IIA) or steps 15-18 while purification using vacuum manifold (see seciotn XI.IIB), placing Plasmid Binding Plate in a new Plasmid Elution Plate (optional not included in the kit). Elution Buffer The Elution Buffer does not contain EDTA, which may interfere with some enzymatic reactions. Recovery of elution volume A reduction in recovered volume relative to the starting volume is normal when eluting plates. The table below presents the typical volume of elute recovered from the initial volume of elution buffer, when following the kit protocol. Elution buffer volume Recovered elution buffer (±5 µl) Centrifuge Vacuum 50 µl 35 µl 25 µl 75 µl 65 µl 50 µl 100 µl 90 µl 80 µl 125 µl 115 µl 95 µl 150 µl 140 µl 120 µl 175 µl 155 µl 145 µl Balance the centrifuge The use of second RNA Binding Plate placed onto a Collection Plate avoids the need to balance the centrifuge. IX. SAMPLE PREPARATION Bacterial culture preparation It is essential to use fresh overnight broth culture in order to obtain high yield and no genomic DNA contaminations. Bacterial culture should be rejuvenated by streaking a single colony on a Luria-Bertani Agar (LA) plate containing suitable a n t i b i o t i c s. A f t e r i n c u b a t i o n, s e l e c t a s i n g l e c o l o n y a n d u s e i t t o i n o c u l a t e www.dnagdansk.com 8
Cat. no. EM21 the LB medium (containing antibiotic) and incubate overnight at 37 C with intense m i x i n g (a p p. 20 0 r p m). I n c u b a t i o n s h o u l d b e t e r m i n a t e d a t a c u l t u r e d e n s i t y of 2.0-5.0 A 600 units per ml (12-16 hours). To provide optimal mixing and aeration conditions, use a flask which is at least four times larger than the culture volume. A baffled culture flask can also be used. If the final concentration of eluted plasmid is not satisfactory we suggest to increase the initial cellular culture volume (maximum 1.5 ml). The optimal isolation parameters are obtained by processing 0.6 ml of culture. In some cases (for example, low density of bacterial cultures, medium and low copy number plasmids), it may be necessary to use larger volumes of bacterial culture. The cellular culture volume, max 1.5 ml, after pelleting must be re-suspended in 600 µl TE buffer or medium. Bacterial Cultures in a 96 deep-well plate Transfer 0.6 ml of fresh LB medium containing aropriate antibiotics into each well of a 96 deep-well plate then inoculate each well with a single bacterial colony. Seal the plate with microporous tape or an adhesive seal. When using non-porous adhesive tape, pierce 2-3 holes in the tape with a needle above each well to promote air exchange. Incubate at 37 C for 20-24 hours with 180-250 rpm shaking. Pellet the bacterial culture in the plate by centrifugation for 5 min at 2.1 k x g. During centrifugation, the plate should be covered with the adhesive seal. Following centrifugation, remove the adhesive seal then remove the supernatant in each well by quickly inverting the plate. X. BEFORE STARTING 1. Mix well each buffer sulied with the kit. Mix the 7x Lysis Buffer very carefully. 2. Ensure that ethanol has been added to the Wash Buffer. If it has not, add aropriate quantity of 96-100% ethanol, the volumes can be found on the bottle labels or in the table given in section II. 3. Examine the buffers. If a sediment has occurred in any of them, incubate it at 37 C (7x Lysis Buffer, Wash Buffer) or mixing occasionally until the sediment has dissolved. Cool to room temperature. 4. Heat a sufficient quantity of the Elution Buffer to 70 C. 5. Unless otherwise stated, conduct all the isolation steps at room temperature. 9
XI. ISOLATION PROTOCOL I. Preparation of lysates: 1. Transfer 600 µl of the fresh overnight broth culture to a well of a 96 deep-well plate (provided by the user) and add 100 µl 7x Lysis Buffer. 2. Mix thoroughly by gently pipetting (5-6 times) or seal tightly the plate with an adhesive seal and gently invert the plate 6-8 times. In order to avoid shearing of the genomic DNA, the sample has to be mixed gently. Do not vortex! After adding the 7x Lysis Buffer, sample should be clear. If not, incubate it at room temperature for 1-2 min. In order to avoid denaturation of the supercoiled plasmid DNA, do not incubate longer than 5 min. 3. Add 350 μl cold Neutralization Buffer to each well. 4. Mix thoroughly by gently pipetting (5-6 times) or seal tightly the plate with a new adhesive seal and gently invert the plate 6-8 times. Sample has to be mixed thoroughly and gently. Do not vortex! Cloudy solution and a white pellet is an effect of protein and genomic DNA precipitation. 5. Centrifuge the 96 deep-well plate at 3k x g for 10 min. II.A Purification using centrifuge: 1. Place the Plasmid Binding Plate onto a Collection Plate (sulied with the kit). 2. Remove the adhesive seal from the 96 deep-well plate (if used) then carefully pipet the supernatant containing the plasmid DNA to each well of the Plasmid Binding Plate using a multichannel piettor. Keep the pipette tip away from the pellet, which contains the genomic DNA and cell remains. Pipette only to a depth of arox. 75% of the volume of each well so as to not disturb the pelleted debris. Unused wells should be covered with the adhesive seal (provided by the user). 3. Centrifuge the Plasmid Binding Plate with Collection Plate at 3k x g for 5-10 min. Discard the flow-through and reuse the Collection Plate. If not all of supernatant passes through the membrane, repeat the centrifugation for 2 min at 3k x g. www.dnagdansk.com 10
Cat. no. EM21 4. Add 600 μl Wash Buffer into each well of the Plasmid Binding Plate and centrifuge for 2 min at minimum 3k x g. Discard the flow-through and reuse the Collection Plate. 5. Add 600 μl Wash Buffer into each well of the Plasmid Binding Plate and centrifuge for 2 min at minimum 3k x g. Discard the flow-through and reuse the Collection Plate. 6. Centrifuge for 20 min at minimum 3k x g or place the Plasmid Binding Plate in an incubator for 10 min at 70 C to evaporate residual alcohol. The wash buffer contains alcohol, which may interfere with some enzymatic reactions and also decrease the elution efficiency. It is therefore vital to remove the alcohol completely from the DNA Binding Plate before elution. To ensure the completely drying of the membrane do not seal the plate. Removal of alcohol by evaporation at 70 C is more efficient than prolonged centrifugation. 7. Discard the Collection Plate and the flow-through and carefully transfer the Plasmid Binding Plate to a sterile Plasmid Elution Plate. 8. Add 50-100 μl Elution Buffer, pre-heated to 70 C, directly onto the center of the membrane in each well. Other buffer volumes in the 20-200 μl range may be used. For instructions, see section VIII. Recommendations and important notes. 9. Incubate the Plasmid Binding Plate at room temperature for 2 min. 10. Centrifuge the stacked plates at 3k x g for 2 min. 11. Remove the Plasmid Binding Plate then seal tightly Plasmid Elution Plate with Elution Adhesive Seal. The isolated DNA is ready for use in downstream alications or for either short-term storage at +4 C or long-term storage at -20 C. 11
XI. ISOLATION PROTOCOL II.B Purification using vacuum manifold: 1. Prepare the vacuum manifold according to manufacturer s instructions. If using an automated liquid handling workstation, prepare the workstation deck as recommended by the manufacturer. 2. Place the Plasmid Binding Plate on top of the manifold. 3. Remove the adhesive seal from the 96 deep-well plate (if used) then carefully pipet the supernatant containing the plasmid DNA to each well of the Plasmid Binding Plate using a multichannel pipettor. Ensure no remains are transferred along with it. Keep the pipette tip away from the pellet, which contains the genomic DNA and cell remains. Pipette only to a depth of arox. 75% of the volume of each well so as to not disturb the pelleted debris. Unused wells should be covered with the adhesive seal (provided by the user). 4. Aly vacuum for 1-2 min until lysate passes through the Plasmid Binding Plate. Release the vacuum. 5. Discard the flow-through and reuse the Collection Plate. 6. Add 600 μl Wash Buffer into each well of the Plasmid Binding Plate. 7. Aly vacuum for 2 min. Release the vacuum. 8. Add 600 μl Wash Buffer into each well of the Plasmid Binding Plate. 9. Aly vacuum for 2 min. Release the vacuum. 10. Place the Plasmid Binding Plate on a stack of paper towels and gently tap to remove residual liquid from the nozzles. Replace the Plasmid Binding Plate on the manifold. www.dnagdansk.com 12
Cat. no. EM21 11. Aply vacuum for 10 min or place the Plasmid Binding Plate in an incubator for 10 min at 70 C to evaporate residual alcohol. Removal of alcohol by evaporation at 70 C is more efficient than prolonged vacuum membrane drying. The wash buffer contains alcohol, which may interfere with some enzymatic reactions and also decrease the elution efficiency. It is therefore vital to remove the alcohol completely from the Plasmid Binding Plate before elution. To ensure the completely drying of the membrane do not seal the plate. 12. Disassemble the manifold to remove the waste tray. Discard the flow-through. 13. Assemble the vacuum manifold with the Plasmid Elution Plate. 14. Place the Plasmid Binding Plate onto the vacuum manifold. 15. Add 50-100 μl Elution Buffer, pre-heated to 70 C, directly onto the center of the membrane in each well. Other buffer volumes in the 20-200 μl range may be used. For instructions, see section VIII. Recommendations and important notes. 16. Incubate the Plasmid Binding Plate at room temperature for 2 min. 17. Aly vacuum for 2 min. Release the vacuum. 18. Disassemble the vacuum manifold to remove the Plasmid Elution Plate, then seal tightly Plasmid Elution Plate with Elution Adhesive Seal. The isolated DNA is ready for use in downstream alications or for either short-term storage at +4 C or long-term storage at -20 C. 13
XII. TROUBLESHOOTING PROBLEM POSSIBLE CAUSE SOLUTION Incomplete cell lysis Plasmid DNA has denatured Low yield of purified DNA Low concentration of purified DNA RNA contamination present Too many cells were taken for DNA purification. Incomplite lysis of bacterial cells. Salt precipitation in 7x Lysis Buffer occurred. The lysate is not clear. Prolonged incubation with the 7x Lysis Buffer. Starting material contained few bacterial cells. Old bacterial culture was taken for DNA isolation. The bacterial cells do not contain plasmids. Incomplete cell lysis. Incomplete transfer of the lysate into a purification minicolumn. Ethanol was not added to the Wash Buffer. The ph of the water used for elution is lower than 7.0. Too much Elution Buffer was used. Improper storage of Neutralization Buffer. The bacterial culture should be at a density of A 600 5.0. For recommended sample volumes, see section IX. Sample preparation. Exceed the time of bacterial cells incubation/lysis in 7x Lysis Buffer up to 5 min, mixing the lysate gently throughout the lysis step. When 7x Lysis Buffer is stored below 20⁰C, a salt precipitation may occur. Redissolve any precipitate by warming the solution at 37⁰C, then mix well and cool down to the room temperature before use. Incubate the lysate at room temperature for 1-2 min. Do not incubate for longer than 5 min to avoid denaturation of supercoiled plasmid DNA. Do not incubate the sample for longer than 5 minutes before adding the Neutralization Buffer. Increase the amount of the starting material. For instructions, see section IX. Sample preparation. Culture cells in a broth medium containing antibiotic for no longer than 16 h. Ensure that the aropriate antibiotics were added to any culture medium used. See Incomplete cell lysis. The clear supernatant can be poured directly into a purification column, however it is the most efficient to transfer the lysate by a pipette. Ensure that 96-100% ethanol was added to the Wash Buffer before use. Use Elution Buffer for DNA elution. Decrease the volume of the Elution Buffer. For details, see section VIII. Recommendations and important notes. The Neutralization Buffer contains RNase A and must be stored at +4 C. www.dnagdansk.com 14
Cat. no. EM21 Isolated DNA is of poor purity Genomic DNA contamination present Inhibition of downstream enzymatic reactions Old bacterial culture has been processed. The culture medium was not removed completely from the cell pellet. One of the washing steps was omitted. The purified DNA contains residual alcohol. Old bacterial culture has been processed. Fragmentation of genomic DNA during cell lysis. The plasmid DNA has denatured. The purified DNA contains residual alcohol. Culture cells in broth medium containing antibiotic for no longer than 16 h. Some medium components may affect DNA purity. The LB medium is recommended for direct culture lysis. If another medium is used, the pellet should be suspended in water or TE buffer prior to lysis. Ensure complete removal of the culture medium from over the pellet. Repeat the isolation, performing both washing steps. Repeat the isolation, giving particular attention to ensuring that no residual Wash Buffer is left in the purification minicolumn after step 6 (Purification using centrifuge XI.IIA) or step 11 (Purification using vacuum manifold XI.IIB). Culture cells in broth medium containing antibiotic for no longer than 16 h. Do not vortex sample when the 7x Lysis Buffer has been added. It may cause the genomic DNA fragmentation and contamination of purified plasmid DNA sample. See Plasmid DNA has denatured. Repeat the isolation, giving particular attention to ensuring that no residual Wash Buffer is left in the purification minicolumn after step 6 (Purification using centrifuge XI.IIA) or step 11 (Purification using vacuum manifold XI.IIB). XIII. SAFETY INFORMATION Neutralization Buffer Caution H302, H315, H319 P305+P351+P338, P302+P352 7x Lysis Buffer Hazard H315, H319 P280, P305+P351+P338 H302 Harmful if swallowed. H315 Causes skin irritation. H319 Causes serious eye irritation. P280 Wear protective gloves/protective clothing/eye protection/face protection. P305 + P351 + P338 IF IN EYES: Rinse continuously with water for several minutes. Remove contact lenses if present and easy to do continue rinsing. P302+P352 IF ON SKIN: Wash with plenty of soap and water.
www.dnagdansk.com