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View additional product information for PureLink™ Quick Gel Extraction Kit - FAQs (K210025, K210012)
23 product FAQs found
以下是为您提供的一些建议：
•许多酶，包括限制性内切酶和连接酶，可被少量琼脂糖和高氯酸盐污染物抑制。正常情况下，这并不会对实验造成影响。但是，如果出现这样的问题，您可使用未经再纯化的DNA，通过增加消化或连接过程中酶的用量或通过延长消化或连接的孵育时间优化实验。另外，也可以在使用相同的反应体系和相同用量的限制性内切酶的情况下，减少消化或连接反应的DNA量。
•洗脱片段中可能有残留乙醇。一定要完全离心以去除洗涤缓冲液并丢弃，使用新试管收集洗脱的DNA。在对乙醇非常敏感的应用中，应将吸附柱打开后，在室温下直立放置15分钟，使多余的乙醇挥发。
•洗涤步骤可能未达到理想效果。在这种情况下，洗脱液中可能含有痕量高氯酸盐。为避免这种情况发生，可将离心时间延长至5分钟，并使用洗涤缓冲液洗2次。
•如果您使用了高浓度琼脂糖或向柱子中加入超过250 mg琼脂糖，应确保执行备选的洗涤步骤（可参考QC protocol）。在对EDTA敏感的应用中，应使用水（pH 7.5–8.5）或无EDTA的10 mM Tris（pH 8.0）洗脱。
常规和低熔点琼脂糖凝胶均适用。当凝胶浓度超过2%时，每100 mg 胶使用600 μL溶胶液进行溶解。
可以，用水洗脱是可以的，不过请确保水质洁净，pH介于7.5和8.5之间。
可以，我们的凝胶提取试剂盒和TAE及TBE琼脂糖凝胶均兼容。
Here are some suggestions for your experiments:
- Many enzymes, including restriction endonucleases and ligases, are inhibited by small amounts of agarose and by perchlorate contaminants. This is not normally a problem. If it is, however, you can use the DNA without repurification by increasing the amount of enzyme used in the digest or ligation or by increasing the digestion incubation time. Also, you may use less DNA in your digest or ligation with the same total volume and the same amount of restriction enzyme.
- There may have been residual ethanol in the eluted fragment. Be sure to thoroughly centrifuge to remove the wash buffer, discard the wash buffer, and use a fresh tube to collect the eluted DNA. For applications that are very sensitive to ethanol, let the open spin column stand for 15 minutes at room temperature to let any excess ethanol evaporate.
- The washing steps may not be as efficient as they should be. Under these circumstances, there may be trace amounts of perchlorate in the eluate. To avoid this, extend the centrifugation times to 5 minutes and wash 2 times with wash buffer.
- Be sure to perform the optional wash step if you are using higher concentrations of agarose or are adding more than 250 mg to the cartridge. If applications are sensitive to EDTA, elute with water (pH 7.5-8.5), or with 10 mM Tris, pH 8.0 without EDTA.
Both regular and low-melting agaroses can be used. When the agarose concentration is above 2%, use 600 µL of solubilization buffer for each 100 mg of gel.
Yes, water may be used, but please ensure that the water is clean and the pH of the water is between 7.5 and 8.5.
Yes, both TAE and TBE agarose gels are compatible with our gel extraction kits.
The DNA pellet from precipitation with isopropanol is easily dislodged when washing with 70% ethanol. It is best to remove the isopropanol supernatant and the ethanol wash by pipetting. Be careful not to shoot the washing buffer directly onto the pellet. Instead, allow the washing buffer to run over the pellet. Regardless of which manufacturer's miniprep kit you use, washing the pellet can be challenging because it is so small.
Multiple bands can occur when the DNA is partially denatured on the gel due to high heat generated during electrophoresis or running the gel too fast. This can appear as multiple bands when the eluted DNA is analyzed on a gel. Denaturation can also occur in AT-rich DNA during the 50 degrees C incubation to dissolve the gel slices. If this happens, solubilize the gel at 37 degrees C for 20 to 30 minutes with repeated vortexing.
There are several reasons why enzymatic reactions can be inhibited:
-Contaminants carried over from the gel purification such as agarose or perchlorate can have inhibitory effects on many enzymes including restriction endonucleases and ligases. This is not normally a problem but if it is, you can use the DNA without re-purification by increasing the amount of enzyme used in the digest or ligation or by increasing the digestion incubation time. Also, you may use less DNA in your digest/ligation with the same total volume and the same amount of restriction enzyme.
-There may have been residual ethanol in the eluted fragment. Be sure to thoroughly centrifuge the column to remove the Wash Buffer and use a fresh tube to collect the eluted DNA. For applications that are very sensitive to ethanol, let the open spin column stand for 15 minutes at room temperature to let any excess ethanol evaporate.
-The washing steps may not be as efficient as they should be. Under these circumstances, there may be trace amounts of perchlorate in the eluate. To avoid this, extend the centrifugation times to 5 minutes and wash 2 times with Wash Buffer.
-Be sure to perform the optional wash step if you are using higher concentrations of agarose and when you are adding more than 250 mg to the cartridge.
-If applications are sensitive to EDTA, elute with water (pH 7.5 to 8.5), or with 10 mM Tris, pH 8.0 without EDTA.
Here are possible reasons:
-Loading more than 400 mg of agarose will decrease the performance of the cartridge.
-An incorrect ratio of Solubilization Buffer to gel was used. Use 30 µL of buffer per 10 mg of gel when the agarose is less than 2%. Use 60 µL of buffer per 10 mg of gel when the agarose is above 2%.
-Ethanol was not added to the Wash Buffer. Ethanol is necessary to keep DNA bound to the silica membrane.
-The bottle with buffer W9 was not kept tightly closed when not in use. Evaporation reduces the ethanol content in buffer W9, resulting in increasingly poor recovery of DNA.
-The gel was not completely solubilized before it was added to the column. Make sure to dissolve at 50 degrees C and to mix every three minutes.
-The DNA was not completely eluted. TE buffer prewarmed to 65 to 70 degrees C can increase elution yield.
-The DNA was supercoiled. Supercoiled DNA will not be eluted from the membrane using the Wash Buffer from the kit.
-The kit was not stored at 4 degrees C. The kit should be stored and used at room temperature. Use of cold buffers may reduce yield.
This can occur for several reasons:
-The incorrect ratio of Gel Solubilization Buffer to agarose mass was used. When the agarose concentration is below 2%, use 300 µL of Gel Solubilization Buffer for every 100 mg of gel. When the agarose concentration is above 2%, use 600 µL of Gel Solubilization Buffer for every 100 mg of gel.
-The solubilization step was carried out below 50 degrees C.
-The sample was not vortexed thoroughly every three minutes.
-The solubilization step was not carried out long enough.
-Was the mass of the agarose gel slice greater than 100 mg? If so, mincing the agarose will accelerate the solubilization.
Supercoiled DNA will not be eluted from the membrane using the standard Wash Buffer that contains 70% ethanol. Supercoiled DNA can be eluted from the membrane only when the Wash Buffer contains 50-55% ethanol.
Yes, but be sure that you are using highly pure water and that the pH of the water is pH 7.5 to 8.5
The agarose gel must be thoroughly dissolved before processing. Frequent and vigorous mixing is critical during the dissolution. Also be sure to use sufficient amounts of Buffer GS1 to dissolve the gel. Mincing of the gel is not generally required but will speed the dissolution process.
A precipitate may form if your sample contains SDS or potassium ions. After solubilization is complete, remove the precipitate by a quick spin and transfer the supernatant to the spin column.
DNA fragments ranging in size from 40 bp to 10 kb can be purified with the PureLink Quick Gel Extraction kit. However, recovery of fragments less than 80 bp will be reduced by 50% or more. For single-stranded DNA and RNA, the lower limit is about 200 NT.
The PureLink kit can be used with both TAE and TBE gels, while the S.N.A.P. Gel Purification Kit is recommended for TAE gels only.
No, TBE gels contain borate, which interferes with the sodium iodide solution. Therefore, only TAE gels can be used. We recommend the PureLink Quick Gel Extraction kit for both TAE and TBE agarose gels.
An excess of cells can actually cause a reduction in yield. Yields go up with cell numbers until a critical point is reached; then yields go down. This is why we strongly recommend growing in LB rather than rich media (e.g., TB) for plasmid purification.
One column from the PureLink Quick Gel Extraction Kit (Cat. No. K210012, K210025) can bind and purify up to 15 µg DNA.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
The S.N.A.P. UV-free Gel Purification Kit utilizes crystal violet as a staining reagent, which can be visualized in non-UV light conditions. This can prevent DNA damage that can be caused by the UV light required by normal ethidium bromide staining. DNA fragments recovered using this kit have higher cloning efficiency. In addition, DNA can be visualized under normal light as a thin violet band while the gel is running and excised as soon as bands are sufficiently resolved.