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View additional product information for PureLink™ HiPure Plasmid Filter Midiprep Kit - FAQs (K210015, K210014)
51 product FAQs found
吸光值测定经常遇到的一个问题是样本的浑浊度。（这可能是由于纯化柱内的树脂残留引起的。）如果在比色皿内有不溶物（经常是肉眼不可见的），大部分的UV光将不被吸收而是被散射，导致出现一个假性的高UV吸收值（例如在260或280 nm）。如果你的A260很高，我们建议你检测A320以确定是否在样本内存在树脂。你也可以试试离心或过滤（使用0.2 µm滤膜）你的样本以去除残留的树脂然后重新测定浓度。
是的，我们推荐购买PureLink HiPure BAC缓冲液试剂盒（Cat. No. K210018）。你需要加入的RNase量将少于该试剂盒内R3缓冲液瓶上标明的RNase量。瓶上标明加入5.6mL的RNase A。这对于BAC纯化实验是正确的量，但是，如果你进行的是标准质粒提取，那么应加入1.4 mL RNase A。
HiPure试剂盒可以从DNA中去除包括内源性核酸酶在内的所有蛋白。对于硅膜法的PureLink Quick Plasmid Miniprep试剂盒，我们建议使用洗涤缓冲液W10进行一步额外的洗涤步骤以去除内源性核酸酶。这一溶液与HiPure系统不兼容，不能用于HiPure试剂盒。此外，可以将洗脱后的溶于TE中的DNA在70°C加热10分钟。这样做可以使任何污染的核酸酶发生热失活。
当质粒DNA出现切口和/或永久变性时，就会有额外的条带出现。在电泳时，带有切口（共价键断裂）的质粒DNA比超螺旋DNA的迁移速率慢。少量的此种类型的DNA存在是正常的而且并不影响下游应用。永久变性的DNA比超螺旋质粒迁移更快并且可能不适合下游应用。确保裂解反应不能超过5分钟。
我们有时会观察到这种现象。这些颗粒不会影响DNA的质量。这些颗粒可以通过在12,000 x g离心1分钟去除。
当溶液过冷时通常会出现这种情况。如有必要，可以将溶液短暂加温至37°C直至沉淀溶解。
如果发现DNA污染，那么可能是细胞裂解步骤和中和步骤出现了问题。有可能中和步骤的混合物没有被正确离心，或者在裂解步骤混合样本时过于激烈。
要降低RNA污染可尝试如下建议：
- 将沉淀缓冲液（N3) 的体积提高10%。（这将降低结合到树脂的RNA量。）
- 将洗涤缓冲液（W8）的盐浓度从800 mM NaCl提高到860 mM NaCl，即向每100mL洗涤缓冲液中加入0.35 g NaCl。（这样做将从树脂上洗涤除去更多的RNA）。
（注意：上述每一步改变都能显著降低RNA污染。这些步骤结合起来基本可以除去所有RNA污染。）
- 确保向重悬缓冲液中加入了RNase A并完全溶解。
- PureLink HiPure Filter Midi和Maxi试剂盒可以更彻底的去除细菌裂解物从而降低RNA污染。
- 如果RNA污染仍然存在，可以提高重悬缓冲液中的RNase A浓度。RNase A浓度可以提高至400 µg/mL。这样做在纯化低拷贝质粒或其它单拷贝DNA类型如BAC时尤其有用。
- 当提取低拷贝质粒时由于使用了大量的细菌所以更容易产生RNA污染问题。
排除细菌培养物的问题：
- 纯化柱中可能加入了过量的细菌。确认细菌培养达到了合适的OD值并且所使用的体积符合试剂盒的规格。确认所使用的培养基，因为TB或高营养培养基会产生高密度菌液从而将导致裂解不完全。
- 确认加入的菌液和缓冲液的体积（用RNase A重悬时，裂解以及中和步骤）。检查加入重悬缓冲液的RNase量。对于高密度或大体积的菌液，可以加入额外量的RNase A至400µg/mL，如前所述。
请检查质粒是否是通过乙醇沉淀并在洗脱后进行了洗涤。如果质粒含盐过多或pH值过高则会引起抑制效应。
可尝试如下建议：
•确保质粒的结合是在室温（RT）下进行的。温度会影响结合溶液的pH值。确保所有其它的溶液也处于室温。
•确认中和之后马上离心时不是在4°C进行。如果是，那么上清必须被平衡至室温然后才能用于上柱结合。我们发现如果裂解产物处于室温则DNA可以更好地和纯化柱的基质结合。
•你可能使用了低拷贝质粒。检查质粒。
•细胞收集步骤的培养基可能去除不完全，因此后续步骤的pH值受到影响。
•在重悬步骤细胞团块可能没有被完全重悬。
•纯化后的DNA可能在异丙醇沉淀和乙醇洗涤之后被过度干燥。仅需在空气中干燥沉淀。
•在异丙醇沉淀和乙醇洗涤过程中沉淀可能会丢失。小心操作此步骤，因为沉淀较滑。最好使用移液器移除醇溶液而不是将其倒出。
•当进行BAC DNA纯化时（此时洗涤缓冲液W8中的NaCl从0.8 M提升至0.9 M），可能使用了过量的洗涤缓冲液W8。请参阅使用手册的BAC纯化部分。
•试试用加热的洗脱缓冲液洗脱DNA。对于小于10kb的质粒，无需加热步骤。对于10-30kb的质粒，可选择加热（65–70°C），加热后洗脱效率可提高~20%。对于大于30kb的质粒，建议进行加热洗脱，加热后洗脱效率可提高~50%。进行一步额外的洗脱步骤可以将洗脱效率提高10%。
•如果洗脱后的DNA中有一些不溶物，它有可能是树脂颗粒（树脂粉末）。这些不溶物可以通过12,000 x g室温离心1分钟去除。
•你也可以试试在中对数生长期（大约在OD590为0.8–1.0时）时向菌液中加入氯霉素。
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。
A common problem encountered with absorbance measurements is turbidity of samples. (This could be caused by residual resin from the column.) If there is insoluble material in the cuvette (not often detected by the naked eye), much of the UV light is not absorbed but scattered, leading to an artificially high UV absorbance reading (at 260 or 280 nm, for example.) If your A260 is high, we recommend that you check the A320 to determine if there is resin in the sample. You can also try to centrifuge or filter (0.2 µm filter) your sample to remove any resin and then recheck the concentration.
Yes, we would recommend purchasing the PureLink HiPure BAC Buffer Kit (Cat. No. K210018). This kit includes Resuspension Buffer (R3) (250 ml), Lysis Buffer (L7) (250 ml), Precipitation Buffer (N3) (250 ml), and RNase A (20 µg/ml) (5 ml).
You will need to add less RNase A than stated on the bottle label of the R3 buffer in this kit. It says to add 5.6 mL of RNase A. This is the correct amount for the BAC protocol; however, if you are performing standard plasmid isolation, 1.4 mL RNase A should be added.
The HiPure kits should remove all protein from the DNA including endonucleases. For the silica-based PureLink Quick Plasmid Miniprep Kit, we recommend an extra wash with the optional Wash Buffer W10 to remove endonucleases. This solution is not compatible with the HiPure system and should not be used with those kits. Alternatively, heat the eluted DNA in TE for 10 min at 70 degrees C. This should heat-inactivate any contaminating nucleases.
Extra bands can occur when plasmid DNA is nicked and/or permanently denatured. Plasmid DNA that has been nicked (covalently opened) will run slower than supercoiled DNA during electrophoresis. A small amount of this species of DNA is common and is suitable for downstream applications. Permanently denatured DNA will migrate ahead of the supercoiled DNA and may not be suitable for downstream applications. Do not allow the lysis reaction to proceed longer than 5 minutes.
We have seen this on occasion. The particles do not affect quality of the DNA. Remove the particles by performimg a 1 minute centrifugation at 12,000 x g.
This is common if the solution gets too cold. If necessary, warm the solution briefly to 37 degrees C until the precipitate is dissolved.
If contaminating DNA is seen, there may have been problems with the cell lysis and neutralization steps. Perhaps the mixture was not centrifuged appropriately or the sample was mixed too harshly during the lysis step.
To reduce RNA contamination:
- Increase the volume of Precipitation Buffer (N3) by 10%. (This will reduce the amount of RNA binding to the resin.)
- Increase the salt concentration in the Wash Buffer (W8) from 800 mM NaCl to 860 mM NaCl by adding 0.35 g NaCl per 100 ml Wash Buffer. (This will wash more of the RNA off the resin.)
(NOTE: Each of these changes alone reduces the amount of RNA contamination significantly. Together these changes eliminate virtually all RNA).
- Make sure that the RNase A was added and thoroughly dissolved in the resuspension buffer.
- PureLink HiPure Filter Midi and Maxi Kits may help reduce RNA contamination by clearing bacterial lysate more thoroughly.
- If RNA contamination persists, the RNase A concentration in the resuspension buffer can be increased. It can go as high as 400 µg/mL. This is particularly useful when isolating low-copy plasmids or other single-copy DNA forms such as BACs.
- Contaminating RNA tends to be a little more of a problem when low copy number plasmids are purified due to a large number of cells being processed.
To troubleshoot the bacterial culture:
- The columns may have been overloaded with too many cells. Verify that the culture used was grown to the proper OD value and that the volumes used were within kit specifications. Verify the medium used, as TB or very rich broth could produce a very dense culture, which may cause reduced lysis.
- Verify volumes of cells and buffers added (resuspension with RNAse A, lysis, neutralization steps). Check on amount of RNase added to resuspension buffer. For cultures with high density or high volumes, you could add extra RNase A, to 400 µg/mL as mentioned above.
Please check to see whether the plasmid was ethanol precipitated and washed after elution from the column. Inhibition may occur if there is too much salt and/or if the pH is too high.
Here are some suggestions to try:
- Make sure the binding of the plasmid is being done at room temperature (RT). Temperature affects the pH of the binding solution. Make sure all other solutions were also warmed to RT.
- Verify that the centrifugation immediately following the neutralization step was not done at 4 degrees C. If it was, the supernatant MUST be warmed to RT before binding on the column. We find that the DNA binds to the matrix of the columns better if the lysate is at room temperature.
- Low copy number plasmid may have been used. Check plasmid.
- Not all the medium may have been removed at the cell harvesting step, so the pH of the subsequent steps was affected.
- The cell pellet may not have been thoroughly resuspended in the resuspension step.
- Purified DNA may have been overdried after isopropanol precipitation and ethanol wash. Only air-dry the pellet.
- Pellet may have been lost during the isopropanol precipitation and ethanol wash. Be careful at this step, as the pellet tends to be slippery. It is best to pipette off alcohol solutions rather than pour them off.
- When working with BAC DNA (and therefore increasing the NaCl from 0.8 M to 0.9 M in the Wash Buffer W8), an excessive volume of Wash Buffer W8 may have been used. See the BAC prep section in the manual.
- Try elution of DNA with heated elution buffer: For plasmids less than 10 kb, no heating is required. For 10-30 kb, heating (65-70 degrees C) is optional, and may increase elution efficiency by approximately 20%. For plasmids larger than 30 kb, heating is recommended, and may increase elution efficiency by approximately 50%. Perform an additional elution to increase yield by up to 10%.
- If there is some insoluble material in the eluted DNA, it could be resin particles (resin fines). These are inert and can be removed by a centrifugation at 12,000 x g for 1 minute at RT.
- You can also try adding chloramphenicol to the culture at mid-log phase (around OD590 0.8-1.0).
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.
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.
Yes, but water of low pH or poor quality may cause a decrease in yield and DNA quality. The TE buffer provided with the PureLink HiPure Plasmid Purification Kits contains 0.1 mM EDTA, which is compatible with most subsequent reactions.
At this time, for purification of large plasmids (>50 kb), we recommend using the standard PureLink HiPure Plasmid Kits (without the HiPure Filter or Precipitator) for purification of large plasmids. There are protocols in PureLink HiPure Plasmid DNA Purification manual for Bacmid, BAC and cosmid.
The buffer compositions are as follows:
Resuspension Buffer (R3): 50 mM Tris-HCl (pH 8.0), 10 mM EDTA
RNase A: (20 mg/mL in R3)
Lysis Buffer (L7): 200 mM NaOH, 1% SDS (w/v)
Precipitation Buffer (N3): 3.1 M potassium acetate (pH 5.5)
Equilibration Buffer (EQ1): 600 mM NaCl, 100 mM sodium acetate(pH 5.0), 0.15% Triton X-100 (v/v)
Wash Buffer (W8): 800 mM NaCl, 100 mM sodium acetate (pH 5.0)
Elution Buffer (E4): 1.25 M NaCl, 100 mM Tris-HCl (pH 8.5)
Please note that these buffer composition data apply to the PureLink HiPure Plasmid Mini, Midi, Maxi, Mega, and Giga kits.