G CAPSULE. For Extracting Nucleic Acids & Proteins from Agarose Gel. (Cat. # , )

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179PR 02 G-Biosciences A Geno Technology, Inc. (USA) brand name G CAPSULE For Extracting Nucleic Acids & Proteins from Agarose Gel (Cat. # ,
179PR 02 G-Biosciences A Geno Technology, Inc. (USA) brand name G CAPSULE For Extracting Nucleic Acids & Proteins from Agarose Gel (Cat. # , ) think proteins! think G-Biosciences INTRODUCTION... 3 ITEM(S) SUPPLIED... 3 STORAGE CONDITIONS... 3 ADDITIONAL ITEMS NEEDED... 4 PROTOCOL SUMMARY... 4 DNA ELECTROELUTION... 5 IMPORTANT NOTES... 5 I. WASH G CAPSULE COMPONENTS:... 5 II. EXCISING DNA BAND... 5 III. ASSEMBLING THE G CAPSULE... 5 IV. ELECTROELUTION OF DNA... 6 V. RECOVERING THE ELECTROELUTED DNA... 6 DNA ELUTION TIME:... 7 RNA ELECTROELUTION... 8 PROTEIN ELECTROELUTION... 8 FOR THIN PROTEIN GELS... 8 TROUBLESHOOTING... 9 CITATIONS RELATED PRODUCTS Page 2 of 12 INTRODUCTION G CAPSULE is an electroelution tool for rapid recovery of PCR products, DNA fragments and proteins from agarose and polyacrylamide gels. G CAPSULE consists of two main parts (Figure 1), G PICK for picking DNA or protein band from the gel, and G TRAP, which traps the migrating DNA or protein during electroelution. After excising the DNA or protein band from the gel with G PICK, the G CAPSULE assembly is then placed in a gel box and a current applied. Under the influence of an electrical field, DNA and protein molecules migrate toward the positive (+) terminal of the electrophoresis apparatus and captured by the G TRAP and become bound to the membrane. Figure 1: G CAPSULE consists of two parts: G PICK for picking DNA or protein band from the gel, and G TRAP for trapping migrating DNA or protein during electroelution. Electroelution with G CAPSULE is a gentle method that eliminates the risks of damage to DNA or protein samples commonly encountered in use of glass milk, spin columns, binding columns and other popular techniques. The G CAPSULE method is simple to perform and eliminates additional steps involving washing, spinning, heating or precipitation. The recovered DNA is of highest quality for use in molecular manipulations such as ligation, restriction enzyme digestion, sequencing, amplification, random priming, and other enzymatic reactions. ITEM(S) SUPPLIED (Cat. # ) Description Size G CAPSULE 55 Capillary Pipette Tip 55 Pin 55 Plunger 1 STORAGE CONDITIONS The kit is shipped at ambient temperature. Upon arrival, store at room temperature. Stable for 2 years, if stored correctly. Page 3 of 12 ADDITIONAL ITEMS NEEDED Horizontal electrophoresis apparatus TAE buffer for DNA Optional: G CAPSULE Accessory Kit (Cat. # ) Prevents G CAPSULE from floating during electrophoresis (Figure 2). The G CAPSULE Weight is designed to trap G CAPSULE when submerged under buffer and prevents it from floating or changing direction during electroelution. Figure 2: G CAPSULE Weight prevents 1 2 G CAPSULE from floating or moving during electroelution. PROTOCOL SUMMARY I. Washing: Wash G CAPSULE thoroughly in elution buffer. II. Excising & Picking up a Gel Band: Use G PICK to punch out the gel band (Figure 3). III. Assembling G CAPSULE : Fill G TRAP with elution buffer. Assemble G PICK and G TRAP together. Use the plunger to push the gel piece close to the membrane. Fill G PICK with elution buffer. IV. Electroelution: Submerge G CAPSULE in the gel tank and apply desired current for electroelution. V. Recover Sample: Reverse current for seconds. Remove the entire buffer from G PICK. Puncture a small hole in the Collection Port, insert the capillary tip and remove the sample collected against the membrane. Figure 3: G CAPSULE Scheme. Page 4 of 12 DNA ELECTROELUTION Important Notes If you are using G CAPSULE for the first time, we recommend performing a few trial runs on molecular weig ht marker bands to become familiar with the G CAPSULE protocol. The critical steps are removing the free buffer from the G PICK, and removing the eluted sample from the collection port of the G TRAP. Use TAE buffer as elution buffer for DNA samples to ensure that subsequent ligation, restriction or sequencing reactions are successful. TBE buffer is known to inhibit T4 DNA Ligase and may inhibit the enzyme that catalyzes other recombinant reactions. However, TBE and any other buffer of your choice can also be used, if the enzyme inhibition is not a concern. I. Wash G CAPSULE components: 1. Disassemble the G CAPSULE unit and fill the G TRAP with ~ 500µl TAE buffer. Submerge both G PICK and G TRAP in TAE buffer for minutes to wash the components and to equilibrate its membrane. 2. Remove the G PICK and G TRAP from the buffer and wash the membrane of the G TRAP 2 3 times with TAE buffer. 3. Washed G TRAP can be kept in TAE buffer until use. If not used right away, store at 4 C in 0.5% sodium azide solution. 4. Immediately before use, place the G TRAP on a clean surface and fill with TAE buffer or elution buffer of choice until the G TRAP overflows. II. Excising DNA band NOTE: To visualize the DNA bands, stain the gel with ethidium bromide or other suitable fluorescent stain and observe the gel under ultraviolet light. 1. Position the tapered, cutting edge over the DNA band to be excised. 2. Push the G PICK into the soft gel until you hit the bottom hard surface of the gel tray. 3. Remove the gel band by tilting the G PICK away from you and slowly pulling out of the gel. NOTE: If there are still remnants of the DNA band left behind in the gel or you fail to pick up the DNA band properly, simply invert the G PICK and use the other end to pick up the remnant of the band. Alternatively, use a sharp scalpel to trim the remnant of the DNA band and load the trimmed gel pieces into the G PICK. III. ASSEMBLING THE G CAPSULE NOTE: Assemble the G CAPSULE immediately before you are ready for the electroelution. 1. Ensure the G TRAP is filled with elution buffer (See step I 4). If not, fill with elution buffer. 2. Position the G PICK, containing the DNA band, in the G TRAP (see Figure 3A) by inserting the tapered, cutting edge of the G PICK into the non membrane side of the G TRAP. Push the G PCIK until it locks in place. Page 5 of 12 3. Ensure there are no air bubbles trapped in the G TRAP. Remove any air bubbles by pipetting buffer into the G CAPSULE. 4. Using the plunger provided, push the gel piece(s) close to the membrane, so that the gel piece is uniformly in contact with the membrane. NOTE: Do not push the plunger too hard or damage to the membrane may occur resulting in loss of sample. IV. ELECTROELUTION OF DNA 1. Fill the G PICK with elution buffer. NOTE: Make sure there is no air bubbles trapped in the G PICK or between gel pieces. 2. Submerge the assembled G CAPSULE in buffer on top of the gel bed of the electrophoresis box such that the G TRAP is facing the (+) terminal (red color terminal). 3. Add pre chilled electrophoresis buffer to the gel electrophoresis box to cover the G PICK. Remove some buffer if the G CAPSULE floats. OPTIONAL: G CAPSULE Weight (Cat. # ) can be used for holding the G CAPSULE once submerged. NOTE: Make sure the G TRAP is facing the (+) terminal, so that the DNA migrates into the G TRAP. 4. Turn on the power supply to electroelute the DNA. For elution time, consult the Guide For DNA Elution Time. V. RECOVERING THE ELECTROELUTED DNA 1. After electroelution is completed, adjust the voltage to ~120 V. Reverse the polarity of the current for seconds to loosen the DNA from the membrane. 2. Turn off the power supply and remove the G CAPSULE from the gel box. NOTE: Recover the DNA immediately after turning off the power supply in order to avoid diffusion of the eluted DNA. To minimize loss of the eluted DNA, do not separate the G PICK from the G TRAP. 3. Hold the G CAPSULE horizontally between your fingers and insert the provided pipette tip, until you reach the gel piece, then remove all the free TAE buffer from the G PICK (Figure 4). Any buffer left in the G PICK will be extracted with the eluted DNA and will result in a more dilute sample. WARNING: Be careful not to hit or disturb the gel piece in the G CAPSULE. If the gel band is disturbed, the DNA in the vicinity of the membrane may flow backward. NOTE: Removing the free TAE buffer from the G PICK relieves the pressure inside the capsule so that the buffer will not leak and carry away the electroeluted DNA. Once the free buffer is removed, surface tension will keep the electroeluted molecules in the vicinity of the membrane. Page 6 of 12 Figure 4 Figure 5 Figure 6 4. Carefully place the G CAPSULE on a dry surface and puncture a small hole in the membrane of the Colle ction Port with the supplied pin (see Figure 5). WARNING: Do not force the pin downward into the membrane; this will damage the membrane and spill the DNA. 5. Pre set a pipette to 50 60µl, and insert the provided capillary pipette tip through the hole in the membrane and gently push the tip downward (See Figure 6). The capillary tip slides over the cutting edge (slope) of the punch. The tip pushes the wet membrane forward as it moves downward into the collection port. 6. Pipette out the DNA collected near the membrane. WARNING: Do not remove the G PICK as this may lead to sample loss. 7. DNA with molecular weights larger than 4000bp is efficiently eluted and recovered using G CAPSULE. Recovery for 2µg of DNA is generally between 80 90%. The DNA recovered from the G CAPSULE is ready for most enzymatic uses. However, some grades of agarose have sulfate and other enzyme inhibitors as contaminants. If you are in any doubt, it is recommended that you precipitate the DNA by any standard method of your choice. DNA ELUTION TIME: Elution of DNA depends on DNA size, concentration of gel, buffer type and electrical current. It is not possible to provide a universal DNA elution time. The following table is simply a guide and will give you some indication of probable elution time in TAE buffer. Gel (%) DNA Size (bp) Voltage Elution Time (mins) 4, V 5 2 2, V , V 1 23, V 8 9, V 6 6, V 4 2, V 1 23, V 7 9, V 4 6, V 3 Page 7 of 12 NOTE: For appropriate elution time, make test runs with t he appropriate size of DNA fragments. If you are not sure of elution time, progre ss of DNA elution can also be monitored under UV light. Use hand held UV lamps. RNA ELECTROELUTION For RNA use, the G CAPSULE must be made RNase free by treating with DEPC [diethylpyrocarbonate] or other RNase removing agents. We recommend our RNaseOUT (Cat. # ) for removal of RNase. Follow the protocol for DNA electroelution, except at step V.1 reverse the current at 100V for 1 min. PROTEIN ELECTROELUTION G CAPSULE can also be used for the electroelution of 4000Da proteins from polyacrylamide gels for protein sequencing, enzyme assays, raising antibodies and other protein studies. A horizontal electrophoresis tank is required for electroelution. Avoid overheating. If your gel box is not fitted with a cooling device, electroelution should be performed in cold room and at a low current. For protein work, it is strongly recommended that G CAPSULE is first soaked for 30 minutes in an appropriate buffer containing 5mM EDTA and 5% BSA solution. Wash extensively with deionized water to remove free BSA. FOR THIN PROTEIN GELS: Protein gels are often very thin and you might encounter difficulty in picking protein bands into the G PICK. You can overcome this problem by first picking a blank piece of agarose gel. Protein Purification: You can purify protein from both non denaturing polyacrylamide gel and denaturing SDS polyacrylamide gels. Use appropriate buffers for electroelution. Page 8 of 12 TROUBLESHOOTING Problem Band not completely excised DNA recovery is poor Band is wider than G PICK DNA band is crowded by other bands, which may lead to cross contamination Suggestion(s) Turn the G PICK around and use the other open end to pick up the remnants of the band. Or after punching out a band, position the G PICK in the G TRAP and push the gel piece against the membrane with the plunger. Use a scalpel or any sharp instrument to trim the remaining DNA band from the gel. Cut the gel band into small pieces and load the pieces into the G PICK. Perform electroelution as usual. The elution time will be longer. The gel piece was disturbed while removing buffer from the G PICK. Electrical current was not reversed or reverse current was allowed to flow longer than seconds. The membrane was damaged. Elution time was not sufficient or was excessive. DNA was degraded. The G CAPSULE was vertical for a longer period, and backward flow of DNA occurred. Any manipulation or prolonged observation was performed before removing all free buffer from the G PICK. After electroelution, G CAPSULE was placed on an absorbing or wet surface or in a puddle of fluid. Punch out two or more gel pieces from the band, one on top of another. When punching out more than one piece from a gel band. Use the other cutting end of the G PICK and punch out the remnants of the band. Depending on the thickness of the gel, you may be able to punch out 3 10 pieces of gel. Or punch out two or more gel pieces as above. Position the G PICK in the G TRAP and use the plunger to push the gel pieces against the membrane as described. Use a scalpel or any sharp instrument to trim the remaining DNA from the gel and load the pieces into the G PICK. Perform electroelution as usual. The elution time will be longer. Use a scalpel or any sharp instrument to cut out the band you intend to extract. Cut the gel band into small pieces and load the pieces into the G PICK. Electroelute as usual. Elution time will be longer. Page 9 of 12 Gel is too thin, leading to difficulty in picking gel bands with G PICK Gel lanes are too close to each other Want to avoid overheating Elution volume is larger than 35µl Buffer is leaking from the G TRAP G CAPSULE is protected by US Patent # First, pick a blank piece of agarose gel into the G PICK from a prepared 4 5mm thick agarose gel in appropriate buffer. Position the G PICK in the G TRAP and push the blank gel pieces against the membrane with the plunger. Cut the band with a sharp instrument and load into the G PICK. Load samples in alternate sample wells. Alternatively, do not load samples in the wells adjacent to your most critical samples. Before electroelution, replace the buffer in the gel box with pre chilled buffer. Run the electroelution in a cold room. The electrophoresis gel was thicker than 5 6mm. The gel piece was damaged, which increased the dead volume of elution buffer in the G PICK. The entire buffer was not removed from the G PICK. The gel piece was not pushed close to the membrane. This is normal, since G CAPSULE is not watertight and buffer will leak between the plastic parts. Therefore, you must be aware of the following instructions: Assemble G CAPSULE just before use and quickly submerge in the tank buffer. Keep it there until you begin electrical current for electroelution. After electroelution is complete and as soon as you remove G CAPSULE from the buffer, you must first remove all free buffer from G PICK. This will relieve pressure so fluid does not leak and carry DNA with it. Once the free buffer is removed, surface tension will keep the electroeluted DNA in the vicinity of the membrane. Remove drops of buffer around G CAPSULE. Use a pipette or tissue paper. Be careful not to suck inside buffer with tissue paper or pipette tip. Do not place G CAPSULE on an absorbing or wet surface, or in a puddle of fluid; this could create capillary suction through the joining parts of G CAPSULE filled with buffer. After electroelution, you may place G CAPSULE on a dry surface, provided that you have followed the above instructions. Page 10 of 12 CITATIONS 1. Chatterjee, S et al (2012) Acta Biochim Biophys Sin. 259:68 2. Cardi, D et al (2010) J. Biol. Chem. 285: Crosslin, James (2009) HortScience. 44: Li, X. et al (2004) Euro J of Phycology. 39:73 5. Beeson, K. et al (2002) Microbiology. 148: Yeager, M. et al (2001) Circ. Res. 88:2e 7. Robu, M. et al (2001) PNAS. 98: Dudley, E. et al (2001) Microbiology.147: Brezinschek, Hans Peter et al (2000) Int. Immunol. 12: Tanaka, K. et al (1999) Yeast. Vol 15: 11: Van Dodeweerd, A. et al (1999) Genome. 42: Ohashi, Y. et al (1998) J. Clin. Invest. 102: Brezinschek, Hans Peter et al (1997) J. Clin. Invest. 99: 2488 RELATED PRODUCTS Download our Molecular Biology & Protein Purification Handbooks molecular biology handbook/ protein purification handbook/ For other related products, visit our website at or contact us. Last saved: 7/27/2012 CMH Page 11 of 12 Page 12 of 12
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