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Thermo Scientific KingFisher Pure RNA Blood Kit
Thermo Fisher Scientific
Chapter 4
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Storage Conditions and Preparation of the Reagents
Table 4-1. Instructions for the preparation of Wash Buffer 1, Wash Buffer 2,
and Rebinding Buffer. Add the indicated volume of 100% isopropanol or
96−100% ethanol to each bottle.
Wash Buffer 1
Wash Buffer 2
Rebinding Buffer
Concentrated buffer
100 ml
30 ml
25 ml
Ethanol (96−100%)
−
120 ml
−
Isopropanol (100%)
90 ml
−
33.4 ml
Total volume
190 ml
150 ml
58.4 ml
After preparing each solution, mark the bottle to indicate that the step has
been completed. The buffers can be stored at room temperature.
Preparation of the DNase I
Storage Solution
To prepare the DNase I storage solution, add 1100 µl of DNase I Reconstitution
Buffer to each vial of lyophilized DNase I. Incubate at room temperature for
5 min. Occasional gentle rotation of the vial helps to dissolve the DNase I, but
avoid forceful mixing. Store the DNase I storage solution at -20°C. Repeated
freezing and thawing should be avoided.
Preparation of the DNase I
Working Solution
Calculate the amount of DNase I working solution needed. For the purification
of one sample, mix 35 µl of 2 x DNase I Buffer, 1.5 µl Manganese Chloride
Solution, 10 µl of DNase I storage solution, and 23.5 µl of nuclease-free water.
The DNase I working solution should be used immediately after preparation.
Starting Material Handling and Storage
RNA purification from blood is recommended to be carried out on the
day of collection. Fresh blood samples collected in anticoagulants
(ethylenediaminetetraacetic acid (EDTA) or citrate) containing vials are not
recommended to be stored at +4°C for longer than six hours. Aliquots
of fresh blood samples, if frozen immediately, can be stored at -80°C for
several months.
Blood samples collected in PAXgene or Tempus tubes should be stored
according to the manufacturer’s instructions.
