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View additional product information for PureLink™ HiPure Plasmid Filter Maxiprep Kit - FAQs (K210016, K210017)
28 product FAQs found
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.