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.

Cloning Tip – How Not To Expose Inserts To UV During Preparation

As mentioned in a previous post¹ there is a way that you can isolate PCR-amplified inserts from DNA agarose gels without exposure of the insert to UV.

First, create replicates of your insert by amplifying multiple PCR reactions.  I generally prepare between 3-5 replicate reactions to increase my insert yield. Prepare a 1% agarose gel + ethidium bromide. After your PCR goes to completion, cool the reactions to 4 degrees or leave it on ice. Add loading dye and load the reactions onto your gel. In the lanes indicated with the letter “L”, add your DNA ladder. I have used a 100bp ladder as an example.

Run the gel. Once completed, remove the gel from the tank and cut the gel as indicated by the red dashed line. Take the smaller portion of the gel to a dark room and visualize on an open UV box. Take a blade or scalpel and mark the gel by nicking the gel edge just above and below the band of interest (green) as indicated by the asterisks. 

Reassemble the entire gel at your bench and then using the nicks made to the gel and the ladders as a guide, carefully cut out the area where your replicate bands of interest are likely to lie as indicated by the dashed red lines.

Gel purify the gel cut-outs containing your bands of interest (green) and proceed with your cloning protocol.

Western Blotting – Why Are My Gels Not Setting?

This question comes up quite often. It can be frustrating having to work to a tight schedule and when for whatever reason your separating and/or stacking gel decides not to set, plans can be sent into a tailspin. 

Usually when a gel doesn’t set, you just start again, but you will be wondering why it didn’t set in the first place. Some common causes are obvious but some are less so.

Clean Your Plates

Those glass plates really need to be clean. If you have residue left over from the previous western run, it can impact on gel polymerization. The same goes for the combs, they need to be cleaned too.

Just before assembling your plates for pouring, spray and wipe (using lint-free tissues) the plates and combs with 70% ethanol.

APS/TEMED

APS and TEMED are important components of the gel mix as they are required for the polymerization of the acrylamide monomers. APS forms the free radicals needed for the polymerization process while TEMED stabilizes those free radicals to speed up the entire process.

APS goes off very easily. If you have made fresh APS and stored it at 4 degrees, it should be fine for about 1-2 weeks (depending on the frequency of use), provided you have not left it to sit at room temperature for extended periods. APS sitting at room temperature for hours should be thrown out.

Ideally, it is better to make fresh APS for each experiment. Just weigh out what you need for each experiment and reconstitute with water.

If despite using fresh APS you find that your gels are setting very slowly i.e. taking hours instead of minutes, you may want to consider using new TEMED.