Found 1 discussion for this experiment
4 years ago
4 years ago by Ralf Friedmann
I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?
Found 3 matching solutions for this experiment
|- Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample.
- Different samples require different methods to achieve complete disruption.
- Incomplete disruption results in significantly reduced nucleic acid yields.
- Overloading the spin columns significantly reduces nucleic acid yields.
|- Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption.
Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids.
Excessive homogenization, on the other hand, results in shorter genomic DNA fragments.
- The centrifugation temperature should be 20–25ºC.
- Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.
|- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
|- Include DNAse treatment for 15-20min.
- Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.
- Use water to elute the RNA that is warmed to ~60`C.
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