Micro-Elute DNA Clean/Extraction Kit
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1 Micro-Elute DNA Clean/Extraction Kit (PCR Clean-up/Gel Extraction/DNA Clean-Up /Concentration Kit) Cat. # : DP034ME/ DP034ME-300 Size : 50/300 Reactions Store at RT For research use only 1
2 Description: The Micro-Elute DNA Clean/Extraction Kit provides silica matrix with ultra-high binding capacity and unique buffer system to recover DNA in small volumes (as little as 10 μl). This design provides highly concentrated DNA in high yields when processing DNA gel elution, PCR clean-up, or DNA desalting. Features: Four applications (PCR/DNA clean-up, gel extraction, DNA concentration) using same components of a single kit. DNA resolved on agarose gels in both TAE and TBE buffers can be recovered with high yields (> 80%) within 15 min. 80%~95% of applied DNA is recovered from PCR reaction or DNA solution by Clean-Up method. Phenol extraction or alcohol precipitation is not needed. DNA purified is free from salt and macromolecular contaminants. Components of the Kit: DP034ME DP034ME Binding Solution 60 ml 360 ml 2. Silica Matrix 1 ml 6 ml 3. Wash Solution 16 ml (add 64 ml of Ethanol before use) 96 ml (add 384 ml of Ethanol before use) 4. Elution Solution 10 ml 60 ml 5. Spin Filter 50 pcs 150 pcs x 2 6. Collection Tube 50 pcs 150 pcs x 2 Materials to be supplied by the user: 100% Ethanol: For preparing the Wash Solution Water bath or heating block at 60 C 1
3 General Procedure for PCR and DNA Clean-Up: 1. Following PCR amplification or other enzymatic manipulations, transfer the reaction mixture (containing the DNA to be purified) into a clean microcentrifuge tube. 2. Add 3 volumes of Binding Solution to the reaction mixture (e.g. 50 μl reaction mixture, add 150 μl Binding Solution) and vortex briefly to mix. 3. Resuspend Silica Matrix until a homogeneous suspension is obtained. Add 15 μl Silica Matrix to the sample and vortex briefly to mix. * Silica Matrix may clog the micropipet tip. If necessary, cut off the tip with scissors to pipet easily. * The DNA binding capacity of Silica Matrix is ~2 μg DNA/μl silica. For DNA >20 μg, add more silica matrix. 4. Incubate for 5~10 min at room temperature. Vortex 2~3 times during incubation. 5. Centrifuge at top speed (12~14,000 x g) for 2 min and carefully remove ~80% of the the supernatant with a micropipette, without disturbing the silica matrix. (e.g. if the total content is 100 μl, remove ~80 μl supernatant, saving the bottom ~20 μl portion with DNA-bound silica matrix). * Do not leave over 20 μl solution, which may cause the silica to clog between the O-ring and wall of the column. 6. Insert the Spin Filter into a Collection Tube. Resuspend the DNA-bound silica matrix by pipetting, and apply the matrix onto the center of the Spin Filter and centrifuge at top speed for 1 min. * Do not apply the matrix along the wall of the column or on the O-ring above the filter. 7. Add 700 μl of Wash Solution to the Spin Filter and centrifuge at top speed for 1 min. Discard the filtrate. Repeat this step once more. 8. Discard the filtrate and centrifuge at top speed for additional 3~5 min to remove residual trace of ethanol. Transfer the Spin Filter to a new 2
4 microcentrifuge tube. 9. Incubate the Spin Filter in a heat oven (45~60 C) for 5 min to evaporate any residual ethanol. The silica in the Spin Filter should turn white. 10. Add 10~20 μl Elution Solution or H 2 O (ph 7.0~8.5) to the center of the Spin Filter (if traces of silica are found stuck to the wall or O-ring of the column, gently scrap the stuck silica and suspend it in 10 μl Elution Solution by pipetting and load the suspension to the center of the filter) and wait for 1~2 min. Centrifuge at top speed for 2 min to elute the DNA. Store the eluted DNA at -20 C. * The retention volume of the Spin Filter and silica is about 2 μl. Hence, if 5 μl elution buffer is used, half of the DNA will be retained by the filter. General Procedure for gel extraction: 1. Following electrophoresis, cut out the desired DNA band ( 500 mg) from agarose gel in TAE or TBE buffer *Up to 500 mg gel slice can be purified in one column purification. 2. Transfer the gel slice into a 1.5 ml clean microcentrifuge tube and add 2 volumes (or 3 volumes if agarose gel > 2%) of Binding Solution to the gel slice. 3. Resuspend the Silica Matrix until a homogeneous suspension is obtained, add 15 μl silica suspension to the gel slice. * Silica Matrix may clog the micropipet tip. If necessary, cut off the tip with scissors to pipet easily. * The DNA binding capacity of Silica Matrix is ~ 2 μg/μl silica. For DNA >20 μg, add more silica matrix. 4. Incubate at 60 C for 5~15 min or longer until gel slice has completely dissolved. Vortex the tube to mix every 2~3 min during incubation. 5. Centrifuge at top speed (12~14,000 x g) for 2 min and carefully remove ~80% of the supernatant with a micropipette, without disturbing the silica matrix. (e.g. if the total content is 100 μl, remove ~80 μl supernatant, saving the 3
5 bottom 20 μl portion with DNA-bound silica matrix). 6. Insert the Spin Filter into a Collection Tube. Resuspend the DNA-bound silica matrix by pipetting, and apply the matrix onto the center of the Spin Filter and centrifuge at top speed for 1 min. * Do not apply the matrix along the wall of the column or on the O-ring above the filter. 7. Add 700 μl of Wash Solution to the Spin Filter and centrifuge at top speed for 1 min. Discard the filtrate. Repeat this step once more. 8. Discard the filtrate and centrifuge at top speed for additional 3~5 min to remove residual trace of ethanol. Transfer the Spin Filter to a new microcentrifuge tube. 9. Incubate the Spin Filter in a heat oven (45~60 C) for 5 min to evaporate any residual ethanol. The silica in the Filter should turn white. 10. Add 10~20 μl Elution Solution or H 2 O (ph 7.0~8.5) to the center of the filter (if traces of silica are found stuck to the wall or O-ring of the column, gently scrap the stuck silica and suspend it in 10 μl Elution Solution by pipetting and load the suspension to the center of the filter) and wait for 1~2 min. Centrifuge at top speed for 2 min to elute the DNA. Store the eluted DNA at -20 C. * The retention volume of the Spin Filter and silica is about 2 μl. Hence, if 5 μl elution solution is used, half of the DNA will be retained by the filter. 4
6 Troubleshooting Guide Problem Low DNA recovery a) Wash Solution was not prepared properly b) Insufficient / no PCR product c) Gel slice dissolved incompletely d) Inefficient DNA binding Comments and Suggestion Make sure to add ethanol to Wash Solution before use. Estimate DNA recovery by running 10% of PCR product before and after purification on an agarose gel. 1) If using a gel slice larger than 500 mg, divide the gel slice and process the pieces in multiple tubes. 2) Cut larger gel slices into several pieces to accelerate gel dissolution. 3) Extend the incubation time to dissolve the gel slice completely. The DNA binging capacity is about 2 μg / μl silica. Use Silica Matrix in amount appropriate for DNA amount. (For DNA > 20 μg, add more silica matrix into solution). If larger amount of DNA is to be purified or if the volume of the binding reaction is greater than 1.5 ml, increase the incubation time of binding step for an additional 15 min. e) DNA was not eluted properly. 1) For DNA larger than 5 Kb, use preheated Elution Solution (60~70 C) in Step 10. 2) DNA is to be eluted only with a low-salt solution [e.g., Elution Solution (10 mm Tris Cl, ph 8.5) or water]. Elution efficiency is ph dependent. The maximum efficiency is achieved between ph 7.0 and 8.5. When using water for elution, make sure that the ph of water is within this range. 3) Ensure that Elution Solution is added to the 5
7 center of the filter and is completely absorbed. 4) Allow the Elution Solution to incubate in the Spin Filter for longer time. Poor performance in downstream enzymatic applications. a) DNA eluate is contaminated with salt. Do not allow the binding or washing flow-through liquid to come in contact with the bottom of the column following centrifugation steps. b) Residual ethanol contamination c) DNA is denatured Low A 260 /A230 Guanidine isothiocyanate contamination Be sure to centrifuge the column at recommended speed for 3~5 min in Step 8 and incubate the column at 45~60 C in a hot-air oven for 5~10 min to evaporate of the ethanol. Add 1/10~1/5 volume of Elution Solution to the eluate and incubate at room temperature for 5 min. Alternatively, incubate the DNA Solution at 95 C for 2 min and cool down slowly to re-anneal the denatured DNA. 1) In the wash step, repeat the 700 μl of Wash Solution addition and let stand for 1 min after adding the wash solution before centrifugation of sample. 2) If low A 260 /A 230 ratio is a concern, precipitate the DNA with ethanol following standard protocol. 6
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