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查看更多产品信息 PureLink™ 384 Plasmid Purification Plates - FAQs (12263026)
31 个常见问题解答
我们通常建议在LB培养基中培养E. coli至 A600为2或细胞密度约为1 x 109 cells/mL。
是的，请根据以下建议步骤进行：
1.使用通常的哺乳动物细胞裂解程序裂解细胞，例如，碱裂解。请注意，这一步必须轻柔，因为断裂的基因组DNA将和质粒一起被纯化出来。
2.使用乙醇或70–80%异丙醇沉淀DNA。这是一个粗沉淀的步骤。
3.将上述沉淀物重悬于600 mM NaCl, 100 mM NaOAC, pH5.0的缓冲液中。
4.将上述溶液加入平衡后的HiPure纯化柱中。
上述步骤适用于提取最大100kb的质粒。需注意的是线粒体DNA也同时被纯化出来。
1.革兰氏阳性菌的裂解条件变化很大，所以你可能需要使用一个已知适用于你所使用的细菌的特殊裂解步骤。试剂盒中提供的裂解材料可能不适合。
2.使用特殊方法裂解后，用70%乙醇或异丙醇对粗提的质粒DNA进行沉淀。确保除去基因组DNA（根据包装内的说明卡片上的提示进行，任何时候都不要进行涡旋混匀）。
3.将离心后的核酸沉淀重悬于较小体积的600 mM NaCl, 100 mM乙酸钠, pH 5.0的缓冲液中（1 mL/mini, 10 mL/ midi, 24 mL/maxi）。
将上述溶液加入平衡后的纯化柱中，根据标准流程继续完成后续步骤。
是的，请参阅以下详细步骤：
1.使用2 mL (mini)/10 mL (midi)/30 mL (maxi) EQ1缓冲液平衡纯化柱。
2.收集噬菌体裂解物（液体或固体裂解物）并确定准确体积。
3.根据体积，加入30 μL/100 μL/400 μL缓冲液X1至10 mL/50 mL/250 mL 噬菌体裂解物中并在37°C孵育30 min。
X1 = 100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10 mM EDTA, 20 mg/mL RNase A, 6 mg/mL DNase I
4.将第3步中的核酸酶消化产物和2 mL/ 10 mL/50 mL冰预冷的缓冲液X2混合，冰上孵育60min。
X2 = 3 M NaCl, 30% (w/v) polyethyleneglycol (PEG) 6000
5.在>10,000 x g离心10分钟以收集噬菌体颗粒。弃上清。
6.用1 mL (mini)/3 mL (midi)/9 mL (maxi)缓冲液X3重悬噬菌体颗粒。
X3 = 100 mM Tris-HCl (pH 8.0), 25 mM EDTA
7.向噬菌体悬液中加入1 mL (mini)/3 mL (midi)/9 mL (maxi)的缓冲液X4。上下颠倒几次充分混匀并在70°C孵育10 分钟 (mini)或20 分钟 (midi and maxi)以裂解噬菌体颗粒。
X4 = 4% (w/v) SDS
8.向裂解物中加入1 mL (mini)/3 mL (midi)/9 mL (maxi)缓冲液X5，颠倒混匀，在≥13,000 x g室温离心10分钟。收集上清，避免吸取太多颗粒，然后直接将其加入平衡后的纯化柱内（见第1步）。让裂解物依靠重力进入树脂内。
X5 = 3.0 M 乙酸钾 (用乙酸调节至pH 5.5)
9.用2 x 2.5 mL (mini), 2 x 10 mL (midi), 或1 x 60 mL (maxi)缓冲液W8洗柱。
10.用0.9 mL (mini)/5 mL (midi)/15 mL (maxi)缓冲液X6洗脱lambda DNA并加入平衡至室温的0.7倍体积异丙醇沉淀DNA。
11.在≥13,000 x g，4°C离心30分钟。因为lambda DNA很粘，所以如果使用的是固定转角离心机则DNA将扩散至整个试管壁。因此，我们推荐使用水平转子离心机（例如HB-4或HB-6 Sorvall离心机），或者，如果没有此类转子，使用二甲基二氯硅烷硅化的离心管（如Corex）。
离心结束后，用80%乙醇洗涤lambda DNA并短时放置使其干燥。然后将lambda DNA溶解于适当体积的TE或10 mM Tris缓冲液(pH 8.0)中。
PureLink HiPure Mini, Midi, 以及Maxi试剂盒可以纯化最大200 kb的质粒，而PureLink HiPure Mega和Giga试剂盒可以纯化最大150kb的质粒。
Miniprep
过夜菌培养液体积： 1-3 mL
高拷贝质粒大致产量： ≤30 μg
Midiprep
过夜菌培养液体积： 15-25 mL
高拷贝质粒大致产量： 100-350 μg
Maxiprep
过夜菌培养液体积： 100-200 mL
高拷贝质粒大致产量： 500-850 μg
我们不建议降低洗脱缓冲液的体积，因为这将导致产量下降。你可以进行一次额外的洗脱以提高产量。
对于任何硅膜柱，使用水进行洗脱通常都是可行的。使用缓冲液的优点是吸光度的读值会比较稳定和精确，因为纯水的pH值可能会很低（pH4-5）。
是的，PureLink HiPure Plasmid试剂盒可以提取BAC DNA, bacmid DNA, cosmid DNA, 或M13 ssDNA。请参阅使用手册获取详细的步骤。
所有被称为“HiPure”的试剂盒均使用离子交换树脂以分离高质粒的质粒DNA，适用于进行转染。Filter试剂盒包含一个滤器以去除细菌裂解物而无需离心，而HiPure FP包含一个滤器和一个沉淀器，从而在去除细菌裂解物和进行DNA沉淀时均无需离心。
是的，我们提供EveryPrep通用多头抽真空装置（Cat. No. K2111-01），可以使用ChargeSwitch Pro Filter Plasmid Mini, Midi, 和Maxi试剂盒直接从该装置上真空洗脱。
离子交换纯化推荐用于需要高纯度和低内毒素水平的实验。硅胶纯化的质粒用于转染不是最佳选择，因为其中有较高水平的内毒素和杂质。离子交换柱对于纯化大分子量的质粒效果也更佳。
260nm吸收值和280nm吸收值的比值（A260/A280）通常被用来判定样本的纯度。对于DNA而言，理想的比值是1.8，但是可以接受的范围为1.7–1.9。A260/A230也经常被用来确定是否存在污染。DNA的A260/A230理想值为1.8–2.0。DNA的纯度也可以用凝胶电泳检测。对于质粒DNA而言，应出现一条明显的单一条带（可能会出现几条额外条带，代表质粒分子的多种形式）。对于基因组DNA，查看高的平均片段的大小。
We typically recommend growing E. coli up to an optical density of 2.0 at 600 nm or a cell density of approximately 1 x 10^9 cells/mL in LB broth.
Yes, please follow our suggested protocol:
1) Lyse mammalian cells with a common mammalian lysis procedure, for instance, alkaline lysis. Please note, be careful to be gentle in this step, as sheared genomic DNA will copurify with plasmid.
2) Precipitate DNA with ethanol or 70-80% isopropanol. This is a crude precipitate.
3) Resuspend in 600 mM NaCl, 100 mM NaOAC, pH adjusted to 5.0.
4) Load onto equilibrated HiPure column.
This should work for plasmids up to 100 kb. Note that mitochondrial DNA will also copurify.
1.Lysis conditions vary greatly for gram-positive bacteria, so you would need to start with a specific lysis protocol that is known to work for the particular bacteria you are working with. The lysis materials and conditions in the kit may not work well.
2.After lysis using the specific protocol, precipitate the crude plasmid DNA with either 70% ethanol or isopropanol. Be sure that the genomic DNA is removed (follow precautions in the package insert; do not vortex at any time).
3.Resuspend the resulting nucleic acid pellet in a small volume (1 mL for mini, 10 mL for midi, 24 mL for maxi) of 600 mM NaCl, 100 mM sodium acetate, pH 5.0.
Apply this solution to an equilibrated column and continue with the standard protocol.
Yes, please see the detailed protocol below:
1.Equilibrate a column with 2 mL (mini)/10 mL (midi)/30 mL (maxi) of buffer EQ1.
2.Collect the phage lysate (liquid or plate lysis) and determine the exact volume.
3.According to the scale, add 30 µL/100 µL/400 µL of buffer X1 to 10 mL/50 mL/250 mL phage lysate and incubate at 37 degrees C for 30 min.
X1 = 100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10 mM EDTA, 20 mg/mL RNase A, 6 mg/mL DNase I
4.Mix the nuclease digest from step 3 with 2 mL/ 10 mL/50 mL ice-cold buffer X2 and incubate on ice for 60 min.
X2 = 3 M NaCl, 30% (w/v) polyethyleneglycol (PEG) 6000
5.To collect the phage particles, centrifuge for 10 minutes at greater than 10,000 x g. Discard the supernatant.
6.Resuspend the pelleted phage particles in 1 mL (mini)/3 mL (midi)/9 mL (maxi) buffer X3 with a pipet.
X3 = 100 mM Tris-HCl (pH 8.0), 25 mM EDTA
7.Add 1 mL (mini)/3 mL (midi)/9 mL (maxi) of buffer X4 to the phage suspension. Mix thoroughly by inverting the tube several times and incubate for 10 minutes (mini) or 20 minutes (midi and maxi) at 70 degrees C to lyse the phage particles.
X4 = 4% (w/v) SDS
8.Add 1 mL (mini)/3 mL (midi)/9 mL (maxi) of buffer X5 to the lysate, mix thoroughly by inverting, and centrifuge for 10 minutes at room temperature and ?13,000 x g. Collect the supernatant without taking too many particles and apply it directly onto the equilibrated column (see step 1). Allow the lysate to enter the resin by gravity flow.
X5 = 3.0 M potassium acetate (pH 5.5 with acetic acid)
9.Wash the column with 2 x 2.5 mL (mini), 2 x 10 mL (midi), or 1 x 60 mL (maxi) of buffer W8.
10.Elute the lambda DNA from the column with 0.9 mL (mini)/5 mL (midi)/15 mL (maxi) of buffer X6 and precipitate the DNA by adding 0.7 volumes of isopropanol, previously equilibrated to room temperature.
X6 = 100 mM sodium acetate (pH 5.0 with acetic acid), 1,500 mM NaCl
11.Centrifuge the DNA for 30 minutes at ?13,000 x g at 4 degrees C. Because lambda DNA is very sticky, it will spread over the whole wall of the centrifuge tube if a fixed angle rotor is used. Therefore, we recommending the use of a swinging bucket rotor (i.e. HB-4 or HB-6 for Sorvall centrifuges), or, if such a rotor is not available, using centrifuge tubes (i.e., Corex) siliconized with dimethyldichlorosilane.
After centrifugation, was the lambda DNA with 80% ethanol and dry it briefly. Dissolve the lambda DNA in a suitable amount of TE or 10 mM Tris buffer (pH 8.0).
The PureLink HiPure Mini, Midi, and Maxi Kits can purify plasmids up to 200 kb, while the PureLink HiPure Mega and Giga Kits can purify plasmids up to 150 kB.
Miniprep
Overnight bacterial culture volume: 1-3 mL
Approximate yield for high copy plasmids: ≤30 µg
Midiprep
Overnight bacterial culture volume: 15-25 mL
Approximate yield for high copy plasmids: 100-350 µg
Maxiprep
Overnight bacterial culture volume: 100-200 mL
Approximate yield for high copy plasmids: 500-850 µg
We do not recommend decreasing the volume of elution buffer, as this will cause yield to drop. You can try to perform an additional elution to increase yield.
For any silica columns, elution with water is generally possible. However, a buffer is preferred for stability and accuracy of absorbance readings, as pure water can have a very low pH (4 - 5).
Yes, the PureLink HiPure Plasmid kits can isolate BAC DNA, bacmid DNA, cosmid DNA, or M13 ssDNA. See the manual for a detailed protocol.
All kits termed ‘HiPure' use anion exchange resin columns to isolate the highest quality plasmid DNA, suitable for transfection. Filter kits include a filter to clear bacterial lysate without centrifugation, while the HiPure FP kits include a filter and precipitator to eliminate the need for centrifugation at either the bacterial lysate clearing or DNA precipitation step.
Yes, we offer our EveryPrep Universal Vacuum Manifold (Cat. No. K2111-01), which allows for direct elution from the manifold using our ChargeSwitch Pro Filter Plasmid Mini, Midi, and Maxi Kits.
Endotoxins are typically any cell-associated bacterial toxins that are part of the outer surface of the cell wall of gram-negative bacteria. Endotoxins can influence cell growth, cell differentiation, contractility, and protein expression in mammalian cells. Endotoxins are released during bacterial lysis, and they can subsequently reduce transfection efficiency and protein expression levels. Please review the following article for more information about endotoxins: Butash KA et al. (2000) Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency. Biotechniques 29(3): 610-614, 616, 618-619.
Anion exchange purification is recommended for higher purity and lower endotoxin levels. Silica-based purification is not optimal for transfection, as there is a higher level of endotoxins and impurities. Larger plasmids also work better with anion exchange columns.
The ratio of absorbance at 260 nm to the absorbance at 280 nm (A260/A280) is typically used to measure purity of the sample. For DNA, the ideal A260/A280 ratio is 1.8, but it can be in the range of 1.7 - 1.9. The A260/A230 ratio is also used to determine if contamination is present. For DNA, the ideal A260/A230 ratio is between 1.8 and 2.0. DNA purity can also be examined by gel analysis. For plasmid DNA, look for a strong, single band (perhaps with a few extra bands representing multimers of the desired molecule). For genomic DNA, look for high average fragment sizes.
No. This product is not recommended for use with low-copy number plasmids when the plasmid DNA is for use in automated fluorescent DNA sequencing. Low-copy number plasmids can be used with this system when the samples are for use in PCR amplification. However, keep in mind that the yields from a low-copy plasmid may not be readily visible on a gel.
No. While the DNA is ideal for automated fluorescent sequencing, PCR, and restriction enzyme digestion, it is not compatible for eukaryotic transfection.
Plasmids up to 28 kb have been isolated and sequenced.
The original QiaRobot does not allow modifications to its protocol; however, more recent models are more flexible. Contact Technical Support to learn about robots that are compatible with the system.