New England Biolabs, T1117L, Monarch® Spin Columns S2D and Tubes

Related Categories Plasmid Purification,, Nucleic Acid Purification Applications Nucleic Acid Purification FAQ 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: I have used 5 ml of cell culture for plasmid isolation with the Monarch® Spin Plasmid Miniprep Kit (NEB #T1110), and I am obtaining low amounts of plasmid and/or contaminating gDNA. Why is this, and what are your suggestions to improve yield and purity? 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 due to an insufficient volume of Monarch Buffer B2 (Lysis Buffer) used in the standard protocol. 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 being harvested after transitioning to the stationary phase, 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: Are the collection tubes part of the Monarch® Spin Column S2D and Tubes (NEB #T1117L) package, or do they have to be bought separately? A: Monarch Spin Column S2D and Tubes (NEB #T1117L) are sold with collection tubes as a set. You will receive 100 spin columns and 100 collection tubes. However, you can also purchase collection tubes only (NEB #T2118). 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.