A PhD student working at the Department of Immunology at the University Medical Center Utrecht shares her positive experience with the NucleoBond Xtra Midi Kit from MACHEREY-NAGEL (MN) for making DNA stocks for retro- and lentiviral transfections.

In our laboratory, we need high-yield DNA isolation for retro- and lentiviral transfections. We need at least 500 μg with a concentration of 1μg/μl. Apart from the yield, the purity of the DNA is also of great importance for doing successful transfections. Our current kit sometimes gives low yields and it is a lengthy process to perform, therefore we were looking for a suitable alternative.

The NucleoBond Xtra Midi Kit from supplier MN was tested alongside a leading Maxi prep kit from company Q for purification of our retroviral plasmid pBullet- CMV-IRES-NEO.

A 400 ml bacterial culture was split into 2 aliquots; 100 ml to be purified using the NucleoBond Xtra Midi Kit and 300 ml for the company Q kit.

There were striking differences in the protocols between both preps (Figure 1), leading to a total time saving of 1.5 hours for the NucleoBond Kit (MN):

• The NucleoBond Xtra Midi Kit eliminates the 20 min ice incubation required by the company Q kit before adding the neutralization buffer
• The column filter from the NucleoBond Xtra Midi Kit removes cell debris without an additional spin step
• The column filtered lysate requires only a single wash in the protocol of the MN kit of the binding column
  

The NucleoBond Xtra Midi Kit protocol was completed in just 2 hours.

Figure 1: Short overview of the NucleoBond Xtra protocol and the differences with the company Q kit			Figure 2: 1,5% agarose gel. Lane 1 Marker, lane 2 Company Q isolated DNA, lane 3 MN isolated DNA. Isolated DNA was cut with NcoI and BamHI. Both isolation methods show the expected pattern (insert band 1550 bp).

 

Prep method	Volume used (ml)	Total yield(µg)	Yield (µg/µl)	A260/280	A260/230	Time
MN NucleoBond Xtra Midi	100	927,9	3,093	1,778	2,175	2 hrs
Company Q kit	300	872,1	2,907	1,827	2,278	3.5 hrs

 

Since we compared a midiprep kit with a maxiprep kit and even added 3x more culture to the maxiprep, we assumed we would obtain a much higher yield with the maxiprep. However, the yields for both kits were similar both in concentration and purity (Table 1). The products were verified by restriction digestion analysis and both produced nice clean bands (Figure 2).

We conclude that the NucleoBond Xtra Midi Kit from MACHEREY-NAGEL is a very good solution for our DNA isolations. I would highly recommend this kit, since its unique design speeds up your DNA isolation procedure and generates very high yields!

Ordering Information

 

Cat. #	Description	Quantity
MN 740410.10/.50/.100	NucleoBond Xtra Midi	10/50/100 preps
MN 740412.10/.50	NucleoBond Xtra Midi Plus	10/50 preps
MN 740420.10/.50	NucleoBond Xtra Midi EF	10/50 preps
MN 740422.10/.50	NucleoBond Xtra Midi Plus EF	10/50 preps
MN 740414.10/.50/.100	NucleoBond Xtra Maxi	10/50/100 preps
MN 740416.10/.50	NucleoBond Xtra Maxi Plus	10/50 preps
MN 740424.10/.50	NucleoBond Xtra Maxi EF	10/50 preps
MN 740426.10/.50	NucleoBond Xtra Maxi Plus EF	10/50 preps
MN 740415	NucleoBond Xtra combi rack	1 rack