DNA Gel Extraction and Purification Using Columns

Spin column purification of DNA inserts from agarose gels is a quick and easy process but the downside is that yields are lower compared to DNA purified using a phenol/chloroform extraction protocol. Notwithstanding this, column purification is quite popular and the yields are sufficient for most applications. Interestingly though, there are times when there appears to be no DNA at the end of it.

Something to take note of is the size cut-off of the columns. If you plan on cloning small to very small inserts, pay attention to the kit you are using because you want to avoid having your inserts fall through the filter. Below is a list of various kits and their minimum size cut-offs:

Company	Kit	Minimum Size (bp)
Agilent Technologies	StrataPrep DNA Gel Extraction Kit	100
Merck Millipore	Montage Gel Extraction Kit	100
GenScript	QuickClean II Gel Extraction Kit	100
Qiagen	QIAquick Gel Extraction Kit	70
Omega Bio-tek	E.Z.N.A.® Gel Extraction Kit	70
Macherey-Nagel	NucleoSpin® Gel and PCR Clean-up	50
Affymetrix	PrepEase® Gel Extraction Kit	50
Zymo Research	Zymoclean™ Gel DNA Recovery Kit	50
NEB	Monarch® DNA Gel Extraction Kit	50
Sigma-Aldrich	GenElute™ Gel Extraction Kit	50
Thermo Fisher Scientific	PureLink® Quick Gel Extraction Kit	40

It goes without saying that if you are trying to clone a 48bp insert, you probably don’t want to be using the QIAquick Gel Extraction Kit.

Graphing Data - How To Calculate Error Bars

When turning numerical data into graphical form (e.g. bar graphs), you sometimes find that the data is much easier to interpret when changes observed in test conditions are presented as fold changes relative to the control, which is set to “1”. In doing this, you might wonder how you would go about calculating the error bars. In the following, I will detail how I go about calculating error bars for relative fold changes:

Below is an example data set with three control and three experimental replicates:

C1. 3.55

C2. 3.82

C3. 3.13

E1. 5.21

E2. 6.74

E3. 5.68

Take the average and you will have:

à C-AVG=3.50     E-AVG=5.88

Next, calculate the relative fold change by dividing by the control average:

à C-AVG=3.50/3.50   E-AVG=5.88/3.50  à  C-RelAVG=1   E-RelAVG=1.68

For the error bars, calculate the SEM:

C-SEM=0.20   E-SEM=0.45

Next calculate the SEM for the relative fold change values by dividing the SEMs with the control average:

à C-SEM=0.20/3.50            E-SEM=0.45/3.50  à  C-RelSEM=0.06   E-RelSEM=0.13

Below is an illustration of the data presented without setting the data relative to the control:

  

Here is the data set relative to the control: