Troubleshooting guide for T2060
| Problem | Common Cause | Suggestions/Solutions |
|---|---|---|
| No RNA purified |
Ethanol was not added to Monarch Buffer WX | Check protocol to ensure correct buffer reconstitution. |
| RNA in flow-through and collection tube after sample loading | Overloaded column | Dispense sample into 3 mg (maximum) aliquots prior to adding Monarch Buffer BX and ethanol. Any pellet in collection tube can be recovered by resuspending with nuclease-free water of the protocol and put through RNA cleanup. |
| Low RNA yield | Reagents added incorrectly | Check protocol to ensure correct buffer reconstitution, order of addition of buffers and ethanol, and proper handling of column flow-through and eluates. |
| Precipitate in binding step was not loaded on the column. | Ensure that entire sample along with all precipitate formed in the binding step after adding Monarch Buffer BX and ethanol is loaded onto the column. | |
| Insufficient mixing of reagents | Ensure the ethanol is thoroughly mixed with RNA sample and Monarch Buffer BX before applying the sample to the RNA Cleanup Column. | |
| Incomplete elution | Ensure appropriate volume of nuclease-free water is used for elution. For RNA ≥ 9 kb, larger elution volumes combined with heated elution will improve yield. Larger elution volumes, multiple elutions, and longer incubation times can increase yield of RNA, but will dilute the sample and may increase processing times. For typical RNA samples, the recommended elution volumes and incubation times should be sufficient. | |
| High degree of RNA secondary structure | Binding and elution of smaller RNAs (< 45 nt) can be affected by secondary structure of the RNA molecules. If poor yield of a small RNA is observed, we recommend diluting your sample with 2 volumes of ethanol instead of one volume in Step 2 of the protocol. | |
| Purified RNA is degraded | RNase contamination | In order to avoid RNase contamination during RNA cleanup, make sure to work on a clean lab bench, wear gloves and use disposable RNase-free pipet tips and microfuge tubes (not provided). Keep all kit components tightly sealed when not in use. |
| Improper storage of RNA | Purified RNA should be used immediately in downstream applications or stored at -70°C. | |
| LowA260/230 ratios | Residual guanidine salt carry-over | Ensure wash steps are carried out prior to eluting sample. During wash steps, the column can be gently inverted 3–4 times after adding wash buffer. Use care to ensure the tip of the column does not contact the flow-through. If unsure, repeat centrifugation. When reusing collection tubes, blot the rim of the tube on a Kimwipe prior to reattachment to the column to remove any residual wash buffer. |
| A 2 ml tube was not used for elution | Ensure that a 2 ml microfuge tube is used for the RNA elution step, to avoid any contact between eluate and the column. Use care to ensure the tip of the column does not contact the flow-through. | |
| Low RNA performance in downstream steps | Salt and/or ethanol carry-over | Ethanol and salt remaining after the washes may inhibit downstream applications. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-centrifuge for 1 minute to ensure traces of salt and ethanol are not carried over in the eluted RNA. |
| DNA contamination | DNA removal may be necessary for certain applications. Incubate RNA sample with DNase I (NEB #M0303), DNase I-XT (NEB #M0570), or Monarch DNase I, Lyophilized (NEB #T2104) and cleanup RNA using the RNA Cleanup Protocol. |
For more troubleshooting and FAQs, please visit the product webpage or reach out to our technical support team at info@neb.com.
