DNA isolation / purification Cells - Primary cells Rat cortical neurons

DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.

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5 years ago

5 years ago by R. Verma India

During centrifugation, the column got clogged and I was unable to continue with the protocol. How can I unclog it?

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Found 3 matching solutions for this experiment

NucleoBond® RNA/DNA

Macherey Nagel

Upstream tips	Protocol tips	Downstream tips
Add ß-mercaptoethanol to the solution and homogenize 3–4 times for each 20 s using a commercial homogenizer (e.g., Polytron, Dounce). Alternatively, other homogenization tools like mortar and pestle in the presence of liquid nitrogen may be used. In order to reduce the viscosity pass the lysate 3 times through a sterile plastic syringe fitted with a 20 gauge needle.

Upstream tips
Add ß-mercaptoethanol to the solution and homogenize 3–4 times for each 20 s using a commercial homogenizer (e.g., Polytron, Dounce). Alternatively, other homogenization tools like mortar and pestle in the presence of liquid nitrogen may be used. In order to reduce the viscosity pass the lysate 3 times through a sterile plastic syringe fitted with a 20 gauge needle.

Manufacturer protocol Publication protocol 1 Relevant paper

DNeasy Blood & Tissue Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Manufacturer protocol Publication protocol 1 Relevant paper

Wizard® Genomic DNA Purification Kit

Promega

Upstream tips	Protocol tips	Downstream tips
	For blood DNA isolation, blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Protocol tips
For blood DNA isolation, blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Manufacturer protocol Publication protocol 1 Relevant paper

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