The Monarch Total RNA Miniprep Kit can be used to extract and purify up to 100 μg of Total RNA from a wide variety of sample types, including cultured cells, blood, and various tissues. Tough-to-lyse samples such as plant, bacteria, and yeast can also be processed with this kit with a few added steps.
The protocol consists of 2 parts:
Sample Disruption and Homogenization, and RNA Binding and Elution.
Because of the kit’s versatility, the protocol for Part 1 differs according to sample type. For simplicity, we have divided this video into chapters to address sample types individually.
Part 1, Chapter 2 Cultured Cells
Part 1, Chapter 3 Mammalian Whole Blood
Part 1, Chapter 4 Tissues and Leukocytes
Part 1, Chapter 5 Tough-to-Lyse Samples (bacteria, plant, yeast)
For details on processing samples previously stored in TRIzol®, Monarch DNA/RNA protection reagent, or RNAlater®, or for instruction on how to use this kit for RNA cleanup, please refer to the protocols in the product manual which can be found at neb.com/t2010.
Part 2 RNA Binding and Elution is almost identical for all sample types, so after viewing your specific protocol Part 1, please tune in to Part 2.
Prepare work space
It is important to work in an environment free from RNases. Always wear gloves, use RNase-free glass and plastic ware, and clean your work areas. We recommend wiping bench tops with a cleaning agent such as RNaseZap.
Next, prepare the following reagents.
Part 1, Chapter 1: Prepare Dnase I, Proteinase K, and RNA Wash Buffer
Reconstitute DNase I by adding nuclease-free water to bring the final concentration to ~1U/μl. For the 50 prep kit, add 275 μl, and for the 5 prep sample kit, add 55 μl of water. Gently invert to mix (do not vortex) and keep on ice until ready to use. For subsequent storage, prepare aliquots and store at -20C.
Reconstitute Proteinase K by adding Proteinase K Resuspension Buffer to bring the final concentration to 20 mg/ml. For the 50 prep kit add 1040 μl and for the 5 prep sample kit add 250 μl. Vortex to mix and keep on ice until ready to use. After use, store at -20C.
Add 4 volumes of Ethanol >95% to the RNA Wash Buffer Concentrate. For the 50 prep kit this will be 100 ml and for the 5 prep sample kit this will be 6.4 ml.
Keep in mind that sample lysis and all subsequent steps of the protocol should be carried out at room temperature and not on ice, this will prevent the formation of precipitates in the buffers.
Use the playlist in the upper left corner to choose sample disruption and homogenization chapter specific to your sample type: cultured cells, blood, tissue, tough-to-lyse (plants, bacteria, yeast).
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