This is a point
where you can safely stop the protocol and store the samples prior to proceeding
to the next step in the protocol.
This caution sign
signifies a step in the protocol that has two paths leading to the same end
point but is dependent on a user variable, like the type of RNA
Starting Material: Total RNA (10 ng–1 μg)
purified mRNA (10–100 ng), or ribosomal depleted total RNA (10–100
The protocol is optimized for approximately 200 bp RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A for recommended fragmentation times and size selection conditions.
Note: Follow steps in Protocol (A) if starting material is
total RNA. Perform mRNA isolation, fragmentation and priming using the NEBNext
Poly (A) mRNA Magnetic Isolation Module (NEB #E7490). If starting material is
purified mRNA or ribosomal depleted RNA, proceed to
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The NEB PCR Cloning Kit allows direct cloning from any amplification reaction, with no purification needed!
The Gibson Assembly™ Cloning Kit is a robust method for assembling 1-2 DNA fragments, with a cloning vector, and ensuring high transformation efficiencies - all in a little less than 2 hours.