Found 1 discussion for this experiment
1 year ago
1 year ago by Ralf Friedmann
I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?
Found 4 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.
- For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).