E.Z.N.A.® Plasmid DNA Mini Kit I, (Q – spin)

What is the final concentration of RNase in Solution I?

100 µg/mL

Can I isolate plasmid larger than 20 kb?

The kits work efficiently for plasmid sizes up to 20 kb. You will see reduced yields for plasmids >20 kb. The recommendations for recovery of plasmids >10 kb – heat the elution buffer to 7 ˚C, incubate the elution buffer on the column for 5 min and do a second elution with the eluate from the first round. We have seen success with recovery of 25 kb plasmids.

Are there any special storage conditions for the components of the plasmid kits?

Solution I (once RNase A is added) should be stored at 2-8 ˚C. All other materials should be stored at room temperature.

Can your plasmid kit work for yeast too?

Yes, following some protocol modifications. You will need to source sorbitol buffer and lyticase.

Please prepare fresh sorbitol buffer (1 M sorbitol; 100 mM sodium EDTA; 14 mM β-mercaptoethanol)

1. Harvest cells (maximum 2 x 10⁷) by centrifuging for 10 minutes at 5000 x g (approximately 7500 rpm). Discard the supernatant.
2. Resuspend the pellet in 600 µL sorbitol buffer and 200 units of lyticase. Incubate at 30 ˚C for 30 minutes.
3. Pellet the spheroblasts by centrifuging for 10 minutes at 300 x g.
4. Continue from step 4 (page 10) of the E.Z.N.A.® Plasmid Mini Kit I manual.

Can the E.Z.N.A. Plasmid Mini Kit I clean-up already eluted plasmid?

Purified plasmid can be re-bound to the HiBind DNA column using HBC Buffer (5 volumes of HBC to 1 elution), followed by 2 rounds of wash with DNA Wash Buffer. The last step is to dry the column and elute the DNA. Note: some DNA will be lost due to the column’s inherent properties.