Troubleshooting Guide for Monarch Spin RNA Isolation Kit (Mini) (NEB #T2110)

Column clogging

Sample input higher than recommended

Reduce amount of starting material to match kit specifications to ensure buffer amounts are sufficient and column is not overloaded. Refer to recommendations under Choosing Input Amounts.

Insufficient Lysis

Increase time of digestion or homogenization.

Centrifuge sample to pellet debris and use only supernatant for next steps.

Use larger volume of buffer for lysis and homogenization.

Low RNA yield

Insufficient Lysis

Increase time of digestion or homogenization.

Centrifuge sample to pellet debris and use only supernatant for next steps.

Use larger volume of buffer for lysis and homogenization.

Sample is degraded

Use RNA preservation reagents to maintain RNA integrity during storage.

Use best practices when working with RNA, harvest samples quickly, flash-freeze samples or preserve in stabilization reagent as soon as possible.

To minimize risk of contaminating RNases, clean surfaces and tools with RNase-removal reagents, always handle samples with appropriate PPE.

See Product Manual under Important Notes Before Starting.

Sample input higher than recommended

Reduce amount of starting material to match kit specifications to ensure buffer amounts are sufficient and column is not overloaded. Overloading leads to inefficient purification, insufficient elution and reduced yield. See Choosing Input Amounts.

Low RNA quality

Sample is degraded

Use RNA preservation reagents to maintain RNA integrity during storage.

Use best practices when working with RNA, harvest samples quickly, flash-freeze samples or preserve in stabilization reagent as soon as possible.

To minimize risk of contaminating RNases, clean surfaces and tools with RNase-removal reagents, always handle samples with appropriate PPE.

Salt/Ethanol carryover

Low A260/230 values indicate residual guanidine salts have been carried over during elution. Ensure wash steps are carried out prior to eluting sample. Do not skip any washes with Buffer BX and Buffer WZ.

Use care to ensure the column tip does not contact the flow-through. If unsure, please repeat centrifugation. When reusing collection tubes, blot rim of tube on a soft paper towel prior to reattachment to the column to remove any residual wash buffer.

Add additional wash step and/or extend spin time for final wash.

Residual protein carryover

Low A260/280 values indicate residual protein in the purified sample. Ensure the Proteinase K step was utilized for the recommended time. Ensure samples have no debris prior to addition of ethanol and loading onto RNA purification column. Do not skip any washes with Buffer BX and Buffer WZ.

DNA contamination

DNA carryover

Perform optional on-column DNase I treatment to remove unwanted gDNA from lysed sample.

Perform in-tube/off-column DNase I treatment to remove gDNA. See Appendix in Product Manual.

Sample input higher than recommended

Reduce amount of starting material to match kit specifications to ensure buffer amounts are sufficient and column is not overloaded.

Low performance of RNA in downstream steps

Salt and/or ethanol carryover has occurred

Use care to ensure the column tip does not contact the flow-through after the final wash. If unsure, please repeat centrifugation.

Be sure to spin the column for 2 minutes following the final wash with Monarch Buffer WZ.

When reusing collection tubes, blot rim of tube on a Kimwipe prior to reattachment to the column to remove any residual wash buffer.

Add additional wash step and/or extend spin time for final wash.

Unusual Spectrophotometric readings

RNA concentration is too low for spectrophotometric analysis

For more concentrated RNA, elute with 10 µl of nuclease-free water.

Increase amount of starting material (within kit specifications). See Choosing Input Amounts or Product Manual.

Silica fines in eluate

Re-spin eluted samples and pipet aliquot from the top of the liquid to ensure the A_260/230 is unaffected by possible elution of silica particles.