Qiagen
RNeasy Midi Kit (50)
75144
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E. coli culture (3 ml) grown in LB medium to an optical density of 2.0 was collected in RNA protect bacteria reagent (6 ml) and pelleted, then used for RNA extraction using Qiagen RNeasy midi-kit. Finally, total RNA was eluted in 200 microliters of RNase free water. Quantity of eluted RNA by Nano-Drop spectrometer was 450 ng/microliter with 260:280 ratio of 2.23; 260:230 ratio of 2.34 and RIN from Agilent bio-analyzer was 9.6. Very high quality total RNA was obtained from using this kit.
Total RNA extraction for RNA-sequencing
3 ml of E. coli culture mixed with 6 ml of RNA protect and then pelleted
Total RNA extraction protocol very simple and easy to follow method. Pellet bacteria after mixing with RNA protect reagent at 1: 2 ratio (Bacteria: RNA protect) and perform the lysis-DNase treatment-washing and elution as per the kit's instructions. Finally elute total RNA from the midi-kit columns using 200 microliter of RNase free water. Check the quantity and quality of extracted RNA by Nano-Drop spectrometer and Agilent Bio-analyzer analysis.
After pelleting bacterial culture, make sure that you remove as much as possible RNA protect and culture supernatent.
Extracted RNA was of high quality with very good yield. A 3 ml culture gave me a yield of total 90 microgram with RIN values hitting above 9.0 consistently and 260:280 ratio were greater than 2.24.
For removal of genomic DNA contamination, perform on-column Qiagen DNase treatment.
Very simple and easy to perform protocol. No hassle of pheolic or chloroform smell.
None
Ideal kit for very good quality RNA extraction for RNA-seq or other down stream applications.
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