S1560 FAQs for polyA Spin mRNA Isolation Kit, RNA Isolation and Detection, Intl, NEB
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polyA Spin mRNA Isolation Kit FAQ

Q1: When using the polyA Spin mRNA Isolation Kit, the columns clogged. How can I correct this?
Q2: When using the polyA Spin mRNA Isolation Kit I obtained a low first-round purity. How can I increase the purity?
Q3: When using the polyA Spin mRNA Isolation Kit I obtained a low yield of mRNA. How can I correct this?

Q1: When using the polyA Spin mRNA Isolation Kit, the columns clogged. How can I correct this?

A1: In cases where a column filter becomes clogged, using a 1 ml micropipette with a sterile tip, transfer the cellulose beads and supernatant to the microcentrifuge tube of clogged spin column. Continue mRNA isolation using "
batch protocol". The protocols are nearly identical, differing only at two steps. It is recommended that the elution be done in a clean spin column (there are 2 additional spin columns provided with each kit). This yields a more concentrated final product.

In cases where further isolations from a particular source that has previously clogged a column are to be done, it may be advisable to shear sample by passing back and forth through a sterile 18 gauge needle or to use a commercially available shearing device. Isolations can also be done using the "batch protocol". This may be done when a total RNA preparation is known to contain large quantities of polysaccharides, genomic DNA, or other such debris. Both methods of isolation are equally facile, with little difference in yields. When doing batch isolations it is recommended that elutions are done using the spin protocol method.


Q2: When using the polyA Spin mRNA Isolation Kit I obtained a low first-round purity. How can I increase the purity?

A2: Check the quality of the total RNA preparation. Preparations containing large quantities of polysaccharides or genomic DNA will lower the purity of isolated poly(A)+ RNA. Isolated poly(A)+ RNA will usually contain small amounts of genomic DNA if present in the initial sample. Poly(A)+ RNA isolated using the polyA Spin Kit is usually 60-80% pure. This is sufficient to make probes for Northerns, translational experiments, subtractive hybridization, or oligo (dT) primed cDNA libraries. Isolated mRNA that will be used to make random primed cDNA libraries should be subjected to a second round of purification.


Q3: When using the polyA Spin mRNA Isolation Kit I obtained a low yield of mRNA. How can I correct this?

A3: Enough mRNA is usually isolated for downstream applications. If the total RNA sample is very small (<100 μg), or the desired messenger RNA is of low abundance, the low salt wash may be omitted. This may lower mRNA puriety, but should serve to increase the yield.