Gel Extraction Mini Spin Column Kit. UltraPrep Gel-Ex. Purification of DNA fragments and plasmids from agarose gels

Transcription

1 Gel Extraction Mini Spin Column Kit UltraPrep Gel-Ex Purification of DNA fragments and plasmids from agarose gels 2006 Molzym, all rights reserved 1 UltraPrep Gel-Ex Manual 01/2006

2 Contents Kit contents 3 Storage and stability 3 Kit description 3 Protocol 4 Troubleshooting 6 Appendix 7 I) DNA precipitation 7 II) Agarose gel electrophoresis of prepared DNA 7 III) Photometric determination of DNA concentration and quality 7 Order information 9 Other Molzym products 10 Molzym GmbH & Co.KG Mary-Astell-Str. 10 D Bremen, Germany Adressenzeile 4 Tel.: +49(0) Fax: +49(0) info@molzym.com Web: 2 UltraPrep Gel-Ex Manual 01/2006

3 Kit contents - UltraPrep Gel-Ex 50 reactions 250 reactions Mini spin columns in 2 ml tubes Collection tubes (1.5 ml) Buffer G1 70 ml 350 ml Buffer G2 10 ml 50 ml Manual 1 1 Included buffers are sufficient for 50x/250x gel extractions, when using up to 300 mg gel slices (agarose concentration 0.8 %) per reaction or 150mg gel slices with 2% agarose concentration. Not included: 2 ml polypropylene tubes Isopropanol 70 % ethanol for washing steps. Storage and Stability The kit shows full performance for at least one year, if stored dry at room temperature (18-25 C). Close bottles immediately after use. Guarantee for full performance of the kit as specified in this manual is only valid if storage conditions are followed. Kit Description This kit contains all ingredients for extraction and purification of DNA fragments and plasmids from agarose gels. The UltraPrep Gel-Ex kit is useful for quick preparation of DNA (up to 20 µg) from TAE-, TBE-, low melting and high melting agarose gels. The extracted DNA is suitable for all downstream applications, including PCR analysis, cloning, labeling, restriction analysis, ligation, transformation, hybridization and sequencing. The range of DNA fragments extractable comprises 200 bp to 12,000 bp. Also, linearized and supercoiled plasmid DNA within a range of 3-12 kb can be extracted. The kit contains spin columns, buffers and reagents necessary for solubilization of agarose gels, DNA binding (except isopropanol) to the matrix and elution of DNA into a small volume from the matrix. Washing of bound DNA is done with 70% ethanol (not included). Each kit contains a manual with detailed protocols for DNA gel extraction and purification. 3 UltraPrep Gel-Ex Manual 01/2006

4 Protocol Important notes: please read before starting! Have 70 % ethanol (v/v) and isopropanol (2-propanol) at hand.! Heat a waterbath to 50 C.! You need a bench top microcentrifuge ( x g, rpm), precision pipettes and sterile pipette tips allowing pipetting volumes up to 100 µl and up to 1000 µl, and sterile 1.5 ml or 2 ml polypropylene tubes. Approximate time: 15 min for up to 4 purifications. Procedure 1. Excise gel slice containing the DNA fragment with a clean scalpel and put it into a 2 ml polypropylene tube (not provided). Minimize the amount of agarose surrounding the DNA fragment. When excising stained DNA fragments under UV light, take care to minimize exposition of DNA to radiation in order to avoid damage. Protect yourself against UV irradiation by wearing protective eye glasses and gloves. 2. Weigh the gel slice. For 2 % agarose gels, add 3 volumes of Buffer G1 to 1 volume of gel slice (10 mg 10 µl). For 2% agarose gels, add 6 volumes of Buffer G1 to 1 volume of gel slice. For example: Add 300 µl buffer G1 to 100 mg gel slice (0.8% agarose). Divide gel slices exceeding 400 mg (or 250 mg of 2% agarose gels) to an additional tube. Caution: Buffer G1 is an irritant. 3. Incubate the tube at 50 C until complete solubilization of the gel (approx. 10 min). For better solubilization vortex the tube every 3 min. 4. Add 1 gel volume of isopropanol into the tube and vortex for 5 sec. 5. Transfer the solution into a spin column. Load up to 800 µl to the column. For sample volumes of more than 800 µl, repeat the loading and centrifugation (see step 6). 6. Centrifuge loaded column at x g ( rpm) for 1 min. Discard flowtrough. 7. Add 500 µl of buffer G1 to the column and centrifuge at x g ( rpm) for 1 min. Discard flow-trough. 8. Wash the column by adding 700 µl of 70% ethanol (not included) and centrifuge at x g ( rpm) for 30 sec. 9. Discard the flow-through and centrifuge the column for additional 2 min at x g ( rpm) to dry the membrane. 4 UltraPrep Gel-Ex Manual 01/2006

5 10. Place the mini spin column into a supplied 1.5 ml polypropylene tube and add µl of buffer G2 (10 mm Tris-HCl, ph 8.0) or sterile, deionized water directly to the center of the membrane. Incubate for 1 min. Finally, centrifuge at x g ( 13,000 rpm) for 1 min to elute the DNA. If you want to elute DNA fragments >10 kb, preheat buffer G2 to 70 C. See page 7 for DNA precipitation (appendix I), analysis of DNA by gel electrophoresis (appendix II) and UV measurement (appendix III). 5 UltraPrep Gel-Ex Manual 01/2006

6 Troubleshooting This guide may help solve problems that may arise. Molzym welcomes comments and suggestions for improvement and supplement of our protocols or any hints on other molecular biology applications. The Molzym team is always pleased to answer any of your questions about our products. Phone: +49 (0) ; Observation Possible cause Suggestions No or low yield of poor elution of DNA Add the elution buffer directly to DNA the center of the mini spin column. Heat buffer G2 or water to 70 C. incomplete gel solubilization Incorrect ratio of buffer G1 to gel slice Take care that the gel is completely solubilized. Ensure that the correct volume of buffer G1 is added to the gel slice. (for agarose gels 2 %, add 30 µl of buffer G1 to each 10 mg of gel. For agarose gels > 2 %, add 60 µl of buffer G1 to each 10 mg of gel). Poor performance of extracted DNA in downstream processes DNA damage by UVirradiation Use UV handlamps while excising gel slices. Minimize exposure of DNA to UV light. 6 UltraPrep Gel-Ex Manual 01/2006

7 Appendix I) DNA precipitation Add sodium acetate (3 M, ph 5.2) to the DNA-containing solution to a final concentration of 0.3 M and mix well. For high recovery of DNA, we recommend the use of glycogen or other commercial precipitation supports. Add 2 volumes of chilled ethanol and mix well. Recover the precipitated DNA by centrifugation (>16,000 x g, 4 C, 15 min). Carefully decant the supernatant and wash the pellet with 70 % ethanol (fill the tube half way and shortly vortex) by centrifugation (>16,000 x g, 4 C, 10 min). Carefully decant the supernatant and air-dry the pellet at room temperature. Dissolve the DNA in the desired volume of 10 mm Tris-HCl, ph 8.0 or deionized, sterile water. II) Agarose gel electrophoresis of prepared DNA A direct method for testing DNA quality in terms of molecular size and conformation (plasmid DNA) is agarose gel electrophoresis. Depending on the amount expected, pipette a 1/10 to 1/50 aliquot of the DNA preparation in a microtube or well of a 96 well plate, fill up with sterile water to 10 or 20 µl and add gel loading buffer (e.g. 0.25% bromophenol blue, 30% glycerol). Mix by pipetting and transfer to a gel slot. Run the gel in usual electrophoresis buffers (e.g. TAE, 40 mm Tris-acetate, 1 mm EDTA, ph 8.0) at 1-5 V/cm. III) Photometric determination of DNA concentration and quality Determination of DNA concentration is done by UV reading at 260 nm and 320 nm (background). Correct measurement is only possible when the DNA is free of RNA and readings are at values between 0.1 and 1 absorption units. DNA preparations should be vortexed shortly and diluted accordingly using 10 mm Tris-HCl or water. As a blank, use buffer EB diluted at the same factor as the DNA sample: DNA concentration (µg/ml) = (A 260nm A 320nm ) x 50 (DNA extinction coefficient) x dilution factor DNA yield (µg) = DNA concentration x sample volume (ml) A standard procedure of measuring DNA quality is the determination of the absorption quotient (Q) of readings at A 260nm and A 280nm : Q = (A 260nm A 320nm )/(A 280nm A 320nm ) For a pure DNA preparation, Q lies between 1.7 and UltraPrep Gel-Ex Manual 01/2006

8 Notes: 8 UltraPrep Gel-Ex Manual 01/2006

9 Order information Mini Spin Column DNA Purification Kit Contents UltraPrep Gel-Ex Purification of DNA 50 mini spin columns, fragments and plasmids reagents, from agarose gels UltraPrep Plasmid Mini Purification of plasmid DNA from 1-6 ml culture 250 mini spin columns, reagents, 50 mini spin columns, reagents, 250 mini spin columns, reagents, Cat. No. D D D D UltraPrep Gel-Ex Manual 01/2006

10 Other Molzym products Mini spin column DNA purification PrestoSpin D Universal Genomic and plasmid DNA PrestoSpin D Lambda Lambda and other bacteriophages PrestoSpin D Bug Gram-positive/Gramnegative bacteria PrestoSpin D Fungi Yeasts and fungi PrestoSpin D Plant Plants and soil PrestoSpin D Food Food/feed of animal or plant origin PrestoSpin D Tissue Tissue including mouse tail PrestoSpin D Blood&Cell Whole blood and cell culture Contents Cat. No. D D mini spin columns, reagents,, D mini spin columns, reagents,, D D D D D D D D D D D D D UltraPrep Gel-Ex Manual 01/2006

11 Mini spin column DNA purification PrestoSpin D Plasmid Midi20H High copy plasmids ; up to 20 ml culture PrestoSpin D Plasmid Midi100L Low copy plasmids; up to 100 ml culture Contents 50 mini spin columns, reagents, 250 mini spin columns, reagents, D D Cat. No. 25 mini spin columns, reagents, D mini spin columns, reagents, D Thermostable DNA polymerases MolTaq For routine PCR MolTaq Red Contains red dye for direct gel loading MolTaq Mastermix dntps, Taq and buffer pre-mixed MolTaq Red Mastermix Red dye-containing mastermix for safe and easy PCR Content 1,000 Units (5 U/µl) 5,000 Units (5 U/µl) 1,000 Units (5 U/µl) 5,000 Units (5 U/µl) 250 reactions 1000 reactions 250 reactions 1000 reactions Cat. No. P P P P P P P P Also visit our homepage for further products and offers. 11 UltraPrep Gel-Ex Manual 01/2006