{"product_id":"neb-t1110s","title":"New England Biolabs, T1110S, Monarch® Spin Plasmid Miniprep Kit","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e Plasmid Purification,, Nucleic Acid Purification  \u003cb\u003eApplications\u003c\/b\u003e Nucleic Acid Purification  \u003cb\u003eFAQ\u003c\/b\u003e Q: Can I purchase Monarch columns and collection tubes separately? A: Yes, columns and collection tubes (NEB #T1117) for the Monarch Spin Plasmid Miniprep Kit are sold separately. Q: Can I use water to elute the DNA when using the Monarch Spin Plasmid Miniprep kit? A: Yes, water can be used to elute DNA from Monarch columns. For maximum elution efficiency, ensure the water is nuclease-free and the pH is between 7-8.5. Milli-Q™ water is often slightly acidic, requiring pH adjustment. If you are storing the DNA long-term, we recommend using the supplied DNA elution buffer, which contains 0.1 mM EDTA and can help inhibit metal-dependent nucleases. Q: For the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110), which culture media do you recommended for growing E.coli? A: We suggest using LB (Luria-Bertani) media to grow E. coli. Rich media such as 2X YT or Terrific Broth can also be used, but typically result in significantly higher cell densities at saturation. Thus, it is necessary to scale back the amount of culture processed using rich media to avoid insufficient alkaline lysis and column overloading due to higher biomass. Q: Which E.coli strain(s) are recommended for use with the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: Most standard laboratory cloning strains can be used as hosts to propagate plasmids for miniprep purification. Suitable strains available from NEB include NEB 10-beta (NEB #C3019), NEB 5-alpha (NEB #C2987), NEB Turbo (NEB #C2984), and NEB Stable (NEB #C3040). It may be beneficial to avoid strains such as HB101 or the JM100 series, which have high amounts of carbohydrates that can interfere with downstream enzymatic steps. Q: What culture conditions do you recommended for optimal performance with the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: Cultures should be inoculated from a single colony and grown in the presence of an appropriate antibiotic for the duration of the culture. Sub-culturing can be performed from starter cultures, but results may vary. Cells should be harvested as the culture transitions from logarithmic to stationary phase to ensure the greatest quantity of plasmid DNA is present with little cell lysis occurring. In routine cloning workflows with medium to high copy plasmids, a single colony can be used to inoculate a 5-10 ml culture grown at 37°C for 12-16 hrs. Processing 1-3 ml of this culture should yield 3-6 μg of a high copy plasmid or 1-2 μg of a medium copy plasmid. Lower copy plasmids will produce lower yields, which can be addressed by processing larger culture volumes with concomitant scaling of the B1-B3 buffers or by concentration of the purified DNA. Q: How can I prevent RNA contamination in DNA samples when using the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: To avoid RNA contamination in your DNA samples when using the new Monarch® Spin Plasmid Miniprep Kit (NEB #T1110), it is important to use RNase A as directed in the protocol. RNase A should be added to the Monarch Buffer B1 (Resuspension buffer, pink) ensuring the correct concentration. Store the buffer at 4°C after adding RNase A. During the plasmid purification process, at the step where Buffer B3 (neutralization buffer, yellow) is added, ensure the sample is well mixed and allow it to incubate for the recommended amount of time to allow RNA degradation. For bacterial culture volumes exceeding 3 ml, increase the centrifugation time after adding the neutralization buffer to 5 minutes to allow more time for the RNase to digest RNA. Q: What is the smallest volume of elution buffer that can be used with Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: The protocol is optimized to provide good yields with elution volume as low as 30 μl of elution buffer. Lower amounts can be used to increase concentration but the overall yield will be reduced due to incomplete wetting of the membrane. Higher elution volumes can also be used, but the eluted DNA will have a lower concentration. Q: What is the composition of each buffer provided with the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: The composition of the buffers is proprietary. We can, however, share the following: Monarch Buffer B1 (Resuspension buffer): Tris-based resuspension buffer Monarch Buffer B2 (Lysis buffer): Sodium hydroxide and SDS-based lysis buffer Monarch Buffer B3 (Neutralization buffer): Guanidine hydrochloride-based neutralization buffer Monarch Buffer BZ (Wash buffer 1): Guanidine\/isopropanol-based wash buffer Monarch Buffer WZ (Wash buffer 2): Ethanol-based wash buffer Monarch Buffer EY (Elution buffer): 10 mM Tris, 0.1 mM EDTA, pH 8.5 elution buffer Q: When using the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110), why might I observe a smear above the plasmid band on an agarose gel? A: The presence of a smear at a higher molecular weight than the plasmid band may indicate that genomic DNA is contaminating the plasmid prep. While this is uncommon, genomic DNA can be sheared during lysis and co-purify with the plasmid. Vigorous shaking or vortexing after adding Buffer B2 will likely produce sheared genomic DNA and should be avoided. If the lysate is very viscous and does not mix well, start with fewer cells for the miniprep. Q: When using the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110), what would explain additional bands I see on the gel? A: The predominant form of DNA produced should be supercoiled, which typically has faster mobility during agarose gel electrophoresis than a linear plasmid. In addition to these two forms, you might also observe: An open, single-stranded form at lower molecular weights than the supercoiled form. This is due to excessive alkaline lysis, leading to denaturing of the plasmid. A nicked or relaxed form, referred to as open circular, typically migrating higher than the linear form. Concatemers or plasmid multimers at even higher molecular weights. The relative positions of these bands may differ in TAE vs. TBE buffers. Q: What factors affect the recovery of plasmid DNA when using the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: There are factors both upstream and during the prep that can affect plasmid recovery. Plasmid copy number, plasmid size, insert toxicity, host strain, antibiotic selection, growth media, and culture conditions can all affect downstream plasmid recovery. Plasmid characteristics: Plasmid copy number, size, and insert toxicity directly affect the amount of plasmid contained in each cell. For higher yields, choose high copy plasmids when appropriate, especially for plasmids ≤ 10kb. Larger plasmids, up to 25 kb, can be isolated, but yields will be reduced. Host strain and culture conditions: The choice of host strain, antibiotic selection, growth media and culture conditions are crucial. Maintaining antibiotic selection during growth ensures that the plasmid is not lost and prevents the culture being overtaken by a faster-growing population of cells without plasmid. Proper aeration and growth temperature ensure cells grow logarithmically with little cell lysis. Preparation procedure: Using the appropriate amount of cells and following the protocol is key. Deviating from the guidance to \"boost yields\" often results in reduced yield and DNA quality. Ensure the bacterial pellet is fully resuspended (no visible clumps) before adding Monarch Buffer B2 for alkaline lysis, which denatures both chromosomal and plasmid DNA. Neutralization: Ensure neutralization is complete after Monarch Buffer B3 addition and that the plasmid DNA has renatured by checking that the solution is completely yellow before centrifugation. Incomplete neutralization may cause the cell debris (protein\/ chromosomal DNA\/ cell membrane) not to be pelleted to the bottom of the tube, making recovery of the supernatant more difficult. Use care to avoid transferring cell debris that may clog the Monarch column. Q: What factors affect my (A260\/280) after performing a plasmid miniprep using the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: During a plasmid miniprep, the A260\/280 ratio indicates the relative purity of the eluted DNA by comparing absorbance at 260nm, where nucleic acids have absorbance maxima, to 280nm, where proteins have absorbance maxima. Inefficient neutralization or insufficient washing may allow the excess protein to remain associated with the matrix and elute with the DNA. Using the supplied elution buffer to elute the sample and blank the spectrophotometer will ensure no shift in the 260\/280 due to sample acidity. If water is used to elute the DNA, use the same water as a blank on the spectrophotometer. Q: What factors affect my (A260\/230) after performing a plasmid miniprep using the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110)? A: Media components and some cellular components can reduce the A260\/A230 ratio. Following the protocol will ensure these contaminants are removed. Additionally, some particulates may elute with the DNA, affecting the ratio. If this is the case, perform an additional 15-second spin of the eluted DNA to settle the particulates before evaluation by spectrometry (Nanodrop®) should help. Withdrawing the DNA sample from the top of the sample can help avoid transferring any particulates. Q: What is the maximum binding capacity of the Monarch Plasmid Miniprep Column? A: The matrix supplied in each column is sufficient to bind 20 micrograms of purified DNA under testing conditions. Performing a miniprep introduces cellular components such as carbohydrate, protein, and residual nucleic acid from the host; all of which can compete for binding to the matrix and affect recovery of plasmid DNA. Q: What should I do differently when using a low copy plasmid? A: Recovery of low-copy plasmids is always reduced in comparison to high copy plasmids. Ensuring the culture is grown with proper aeration and harvested before significant cell lysis occurs will increase the number of intact cells harboring the plasmid. Using more cells can make up some of the deficiency, but stay within the supplied guidelines to ensure alkaline lysis is efficient and the column does not get overloaded with cellular components. Please also review our guidelines for working with low copy plasmids and our application note on removal of genomic DNA when working with low copy plasmids. Q: Do the dyes in the buffer of the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110) affect downstream DNA applications? A: No, the purified DNA has been tested in a variety of applications, including restriction enzyme digestion, DNA sequencing, end-point PCR, and transfection of mammalian cells, and they do not pose any issues for downstream applications. Q: Can I use DNA isolated from the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110) for transfection? A: Yes. We have successfully transfected plasmids prepared with our kit into HEK 293 cell lines using Lipofectamine 3000 reagent, with efficiencies similar to market leading miniprep suppliers. Q: Are Monarch spin columns compatible with Vacuum Manifolds? A: We have validated the columns from the following Monarch kits for use on common benchtop vacuum manifolds: Plasmid Miniprep Kit (NEB #T1010), DNA Gel Extraction Kit (NEB #T1020), PCR \u0026amp; DNA Cleanup Kit (NEB #T1030), and RNA Cleanup Kits (NEB #T2030, #T2040, #T2050). Vacuum manifolds can be utilized with other Monarch columns, but may require optimization and\/or additional time for liquid to completely flow through. Q: Can the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110) guarantee endotoxin-free plasmid DNA recovery? A: Although Monarch Buffer WZ (Wash Buffer 1) does remove a large majority of endotoxin from the sample, the DNA recovered cannot be guaranteed to be completely free of endotoxin. That said, we have successfully used plasmid DNA recovered using our kits to transfect robust cell lines. While other vendors may claim their minipreps kit are “endotoxin free,” there is usually still some measurable endotoxin. Q: Why might low yields of plasmid DNA or high levels of contaminating genomic DNA (gDNA) occur when using 5 ml of cell culture for plasmid isolation with the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110), and how can this be improved? A: A lower yield when using 5 ml cell cultures may indicate that too many cells were used in the plasmid isolation. Though we advise that our kit can accommodate up to 5 ml of culture (≤OD 15), there are some cases in which the density of the cells is too high with this volume. Using too many cells can result in incomplete cell lysis, reduced removal of host gDNA, and lower yield. Additionally, the lysis of too many cells will produce excess cell debris and contaminating gDNA that can overwhelm the column and decrease its capacity for binding the plasmid DNA. If the cell culture is harvested after transitioning to the stationary phase, then using less cell culture volume in the isolation may provide better yields. Splitting the 5 ml culture in half and processing it in two columns will likely produce better results. Alternatively, doubling the resuspension, lysis, and neutralization volumes may allow better column performance but will necessitate loading the column twice, each time with half the neutralized sample. Q: Can components from the older Monarch® Plasmid Miniprep Kit (NEB #T1010) be used interchangeably with those from the new version of the kit (Monarch Spin Plasmid Miniprep Kit, NEB #T1110)? A: Yes, while the new version of the Monarch Spin Plasmid Miniprep Kit is developed with improvements to both the physical column and buffer chemistry, the previous version's components are compatible with the updated kit. The purified plasmid DNA will remain high-quality, suitable for various downstream applications; however, there may be up to 20% variances in yield in some cases. Q: Before use, how should I prepare the buffers? Do I need to add isopropanol and\/or ethanol to the buffers prior to use? A: Yes, isopropanol and ethanol needs to be added to Monarch Buffer BZ and Monarch Buffer WZ, prior to use. Monarch RNase A should be added to Monarch Buffer B1 prior to use, and then the buffer should be stored at 4°C. Please see the Buffer Preparation Guidance (NEB #T1110) with specific instructions for the kits on preparing these buffers for use. Q: I notice that I'm applying force to fit the column into a collection tube. Should I be doing this? What collection tubes are compatible with columns? A: Matching collection tubes are provided in all Monarch® kits for the spin columns. If you use collection tubes outside of a given kit, please use the Monarch® Spin Collection Tubes (NEB #T2118). Do not force any columns into non-recommended collection tubes that have a very tight fit to avoid damaging the column. Q: I see some silica residue in my Monarch column. Is this a problem? A: No, this is not an issue. The presence of some silica residue or particles in the nucleic acid purification column does not impact nucleic acid binding or purity. Our columns are designed to retain silica fibers, helping ensure they do not end up in the final eluate. Q: The enzymes in the kit were not stored at -20°C right away. Will they still work? A: Yes, unopened Monarch RNase A is stable at ambient temperatures short-term. After opening, Monarch RNase A should be stored at -20°C. 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