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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):
|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!
|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|