{"product_id":"neb-t3016l","title":"New England Biolabs, T3016L, Monarch® gDNA Elution Buffer","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e Genomic DNA Extraction \u0026amp; Purification,, Nucleic Acid Purification  \u003cb\u003eApplications\u003c\/b\u003e Nucleic Acid Purification  \u003cb\u003eFAQ\u003c\/b\u003e Q: What is the composition of each buffer provided with the Monarch® Spin gDNA Extraction Kit? A: The compositions of the buffers are proprietary. However, we can share the following information: Monarch® gDNA Tissue Lysis Buffer....................Detergent-based lysis buffer Monarch® gDNA Cell Lysis Buffer........................Guanidinium hydrochloride-based lysis buffer Monarch® gDNA Blood Lysis Buffer.....................Guanidinium hydrochloride-based lysis buffer Monarch® gDNA Binding Buffer...........................Guanidinium thiocyanate-based buffer Monarch® gDNA Wash Buffer..............................Ethanol-based wash buffer Monarch® gDNA Elution Buffer............................10 mM Tris-HCl, pH 9.0, 0.1 mM EDTA Q: What is the minimum elution volume that can be used with the Monarch® Spin gDNA Extraction Kit columns? A: The minimum elution volume is 35 µl. For maximum recovery, 100 µl is recommended. Smaller elution volumes will lead to more concentrated samples but also a lower yield. Typically, when 35 µl is used for elution, yields are approximately 20% lower than when using 100 µl. Using elution volumes lower than 35 µl will result in inconsistent wetting of the silica matrix and significantly reduced recovery. Recovery of Genomic DNA using various elution volumes in the Monarch Spin gDNA Extraction Kit Optimal elution volume range for elution is 35 – 100 µl. A lysate pool was prepared by using 10 mg RNAlater®-stabilized rat liver samples and following the Monarch tissue protocol. Elution volumes used in triplicates samples were 25, 30, 35, 40, 50, 75, and 100 µl. The average yield obtained with 100 µl elution volume was 19.1 µg, which was considered 100% yield. Using 35 µl to elute, the average yield was 81.6%; 25 µl and 20 µl yielded 74% and 70.6%, respectively. For volumes ≥35 µl, the recovered volume after elution was ~5 µl lower than the elution volume added and for \u0026lt;30µl the recovered volume was ~10 µl lower. Tests performed with other starting materials showed similar results, with a 20-25% reduction in overall yield when elution volumes were reduced from 100 to 35 µl. For more information, see “Considerations for Elution and Storage” in the product manual. Q: How important is it to pre-heat the elution buffer to 60°C? Would 56°C be OK? A: The pre-heating of the elution buffer is essential for maximal recovery with the Monarch® Spin gDNA Extraction Kit. If more convenient, a temperature of 56°C can be used but may result in a very slight reduction in yield. Room temperature elution buffer can also be used but will result in a 20-30% reduction in yield and may require a second elution. Additionally, at room temperature, large gDNA fragments will elute less efficiently, so the eluted gDNA will have a smaller average size. For more information, see “Considerations for Elution and Storage” in the product manual. Q: Can I get better recovery with the Monarch® Spin gDNA Extraction Kit if I do a second elution with my eluent from the first elution? A: The Monarch protocol for gDNA purification has been optimized for maximum speed and convenience. The elution step with preheated elution buffer enables maximal recovery in just one elution step. However, a second elution step can be performed to maximize recovery. This second elution can be performed using a fresh aliquot of Monarch gDNA Elution Buffer or the eluent from the first elution, to minimize dilution of the DNA. A 10% increase in the total yield may be achieved. For more information, see “Considerations for Elution and Storage” in the product manual. Q: Can I use a different elution buffer than the one supplied with the Monarch® Spin gDNA Extraction Kit (NEB #T3010)? A: The elution buffer supplied with the kit is 10 mM Tris-HCl, pH 9.0, 0.1 mM EDTA. This buffer is optimal for long term storage of gDNA. Any low-salt buffer can be used for elution, such as 10 mM Tris-HCl pH 8.5, TE Buffer or nuclease-free water. Preheating the elution buffer to 60°C ensures rapid and efficient elution of the gDNA. For more information, see “Considerations for Elution and Storage” in the product manual. Q: What size gDNA can be purified with the Monarch® Spin gDNA Extraction Kit? A: Assuming high quality input material has been used, the peak size of gDNA isolated with this kit ranges from 50-60 kb. Typical DNA Integrity Numbers (DINs) for gDNA isolated from blood, cells or tissues are around 9.0. The Monarch gDNA purification protocol is optimized for isolation of gDNA that is larger than that isolated by other commercial kits. For example, it includes elution with preheated buffer that enhances the elution of larger pieces of DNA. Some starting materials such as buccal swabs or saliva, however, contain a significant proportion of dead cells. In those cells, apoptotic mechanisms have activated nucleases that digest the gDNA to shorter fragments. The peak size of gDNA isolated from such samples is usually in the range 20-40 kb. Q: How can I assess gDNA integrity and purity? A: Purity of eluted gDNA samples is assessed by reviewing OD ratios collected during routine spectrophotometry. Pure gDNA typically has an A260\/280 of 1.8–1.9 and an A260\/230 of 1.8–2.5. The integrity of the gDNA is assessed by loading approximately 100 ng per sample on a 0.75% agarose gel and comparing size distribution to a suitable marker, such as lambda DNA, either as full length DNA (NEB #N3011) or digested with HindIII (NEB #N3012). Typically, for intact gDNA, the majority of the gDNA signal will be larger than the upper band of the HindIII digest. Alternatively, gDNA samples can be run on an Agilent TapeStation® using a Genomic DNA ScreenTape. This system will provide information on the peak size of the gDNA and the overall integrity via the DIN (DNA Integrity Number). High peak sizes (\u0026gt;50 kb) and DINs \u0026gt;8.5 indicate that the DNA is of high quality. For more information on assessing nucleic acid purity, download our application note, A Practical Guide to Analyzing Nucleic Acid Concentration and Purity with Microvolume Spectrophotometers. Q: Can I omit the low-speed binding centrifugation step to save time? A: You may omit this step if you wish and you will still get good results. However, results will be inconsistent, and depending on the starting material, yields may be reduced by 5-30%. Binding at low speed is particularly important for optimal recovery when processing tissue samples containing high amounts of DNA and when carrying out the Genomic DNA Cleanup Protocol. Q: How should I store purified gDNA samples? A: Purified gDNA samples can be stored at 4°C. However, for long term storage, we recommend storing them at -20°C. The Monarch® Spin gDNA Extraction Kit (10 mM Tris-HCl, pH 9.0, 0.1 mM EDTA) that is supplied with the Monarch Genomic DNA Purification Kit provides extra safety for long term storage of gDNA samples. Q: When working with other commercial silica column-based kits, I occasionally see a white precipitate in the eluate. What is this? A: Some silica matrices release silica fibers during the elution step. Although silica fibers generally do not affect most downstream applications, they influence OD measurements and should be removed by centrifugation if reliable spectrophotometric values are needed. Monarch Genomic DNA Purification Columns contain a retaining membrane that prevents silica fibers from passing through, thus eliminating the need to perform additional centrifugation prior to accurate spectrophotometric analysis. Q: Is Monarch-purified gDNA compatible with Next Generation Sequencing (NGS) platforms like PacBio and nanopore? A: Yes. gDNA purified with this kit can be used for various applications on these sequencing platforms. Assuming high quality input material has been used, read lengths of up to 50 kb can be routinely achieved using Monarch purified gDNA from different starting materials. Monarch purified DNA quality is superior to SDS Proteinase K-purified DNA commonly used for Nanopore sequencing 2 µg E. coli genomic DNA extracted using either Monarch or by following a traditional SDS Proteinase K isopropanol protocol was enzymatically fragmented with identical incubation times and used as input DNA for MinION library preparation. Oxford Nanopore Technology’s 1D kit protocol was followed to prepare libraries. Both libraries were run on the MinION, the data collected for 48 hours and base called using Albacore. Monarch-purified DNA performed better on all sequencing metrics, including read length, read quality and total sequencing data collected. The graphs illustrate that Monarch purified data provides a higher percentage of high quality sequencing data than SDS Proteinase K purified DNA. Monarch provides excellent quality starting material for Pacific Biosciences (PacBio) sequencing Bacterial gDNA was isolated from 5x109 E. coli ER2683 cells with pACYC184 plasmid by following the Monarch rapid protocol for Gram- bacteria. 10 µg Monarch-purified gDNA was sheared with a Covaris® g-TUBE, targeting 10 kb fragment size. 5 µg sheared DNA was used for library preparation according to the standard PacBio protocol (without size selection). Samples were sequenced on a Pacific Biosciences RSII sequencer, using the P6\/C4 chemistry, loaded as 10 pM SMRTbell library and data were collected in a 5 hr movie. Total bases (yield): 1208 Mb, Average Polymerase read length: 16,046, Polymerase read quality: 0.85. Average Reads of Insert length: 6,060, Reads of Insert quality: 0.89, Longest read of insert: 43,680. A. Insert lengths indicate that the majority of the reads were in the desired insert size range. B. The polymerase read lengths indicate that the purified DNA was of high quality to allow the enzyme to read the insert several times within the SMRTbell construct. C. The loading evaluation data further illustrates that the purified DNA is of high quality. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835534626985,"sku":"T3016L","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-t3016l","provider":"Iright","version":"1.0","type":"link"}