Ways To Ensure Experimental Consistency #3

Commercial Kits

There are many commercial kits available to streamline and simplify various experimental procedures including DNA/RNA extraction, plasmid preparation, cloning, immunoassays, reporter assays, and so forth.

Commercially available kits are certified for quality and have proven efficacy so long as the instructions for use and handling are adhered to. However, there are a few important points to note in regards to reagent and sample preparation, points that some product manuals may not necessarily provide information on. 

* Ensure that components of buffers and reagents are in solution. With some buffers, you may see precipitates. For example, lysis buffers containing SDS tend to develop white precipitates when the room temperature is low. This is the SDS falling out of solution. In such instances, warm up the buffer in a water bath before use. 

* Ensure correct storage of kit components. Make sure that after opening and using individual kit components that they are stored at the recommended temperature. While a kit may arrive at room temperature, after a buffer is opened it may need to be stored at 4 degrees.

* Note the expiry date of the kit and/or specific components.

* If you are required to prepare a component that is not provided (e.g. addition of 3’ A overhangs to inserts for TOPO cloning) be sure to use reagents that are of a quality and purity compatible with subsequent parts of a kit. 

* Ensure that samples are extracted in a buffer that is compatible with the reagents supplied. A notable example is the Bradford Protein Assay, which is incompatible with some detergents and substances.

Dithiothreitol (DTT) vs Beta-mercaptoethanol (BME)

DTT and BME are reducing agents used for the chemical reduction of disulfide bonds. They are commonly added to SDS-PAGE sample buffers and are often used interchangeably. While both DTT and BME are used to achieve the same purpose in SDS-PAGE, they exhibit different chemical properties.

BME

This is very volatile and readily evaporates from solution. Because of its volatility and toxicity, solutions of BME are often handled in a fume cupboard. The disadvantage of this is that frequent usage will increase the rate of evaporation, leading to a decrease in the concentration of a solution of BME over time.

The issue with this is that the chemical reduction of disulfide bonds within proteins and peptides is an equilibrium reaction where bonds are continually breaking and re-forming. Accordingly, excess BME is required to drive the reaction forward to completion. Reciprocally, insufficient quantities of BME in a given reaction will not adequately reduce all protein disulfide bonds with some bonds undergoing reoxidation.

DTT

This is volatile but not to the extent as BME. Unlike BME, the chemical reaction in reducing disulfide bond linkages within proteins and peptides is not an equilibrium reaction. A disulfide reduction reaction using DTT leads to an irreversible change in the DTT molecule where its straight chain structure is altered to a ring structure. Accordingly, use of DTT will avoid issues of disulfide bond reoxidisation. However, DTT is unstable in solution and must be made fresh each time.

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.

Some Thoughts On Creating Stable Cell-Lines

Creating stable cell-lines can be a straightforward or tricky process. In this post I will share some techniques or quirks, which I found worked when creating stable cell-lines.

* Use cells with low passage number – Thaw out your cell stocks^1,2 and start with “new” cells with low passage number.

* Culture cells in antibiotic-free media – By cutting out penicillin/streptomycin, I found increases in my transfection efficiencies. It also avoids any possibility of the penicillin/streptomycin interfering with the selection antibiotic during the selection process.

* Make the selection media fresh – Have aliquots of your selection antibiotic at a higher concentration and dilute it into fresh media each time you need to do a media change.

* Change the selection media daily – After transfection, change the selection media daily up until you reach the limiting dilution stage. It may seem excessive or wasteful but I have found that by having freshly made selection media and a daily media change ensures that the untransfected cells are effectively killed off.

* Have high concentrations of your antibiotics aliquoted – Freeze-thaws and heat-cool cycles can affect the efficacy of some antibiotics so to avoid any issues calculate the approximate amount that you will need for selection and aliquot small amounts into separate tubes.

TOPO TA Cloning – Adding 3’A Overhangs

The TOPO TA cloning kits for subcloning offer an easy way to subclone effectively, provided you can get it to work for you.  The topoisomerase I in which the kit relies on requires the presence of 3’A overhangs on the DNA inserts in order to catalyze the reaction joining insert to vector. Ironically, the enzymes and other components required to add the 3’A overhangs are not supplied with the kits and the protocol provided in the instruction manual is not what I consider ideal.

I have personally never followed the 3’A overhang procedure set out in the product manuals provided; instead, I used my own, which I believe works out more efficiently.

The following is a quick and general run-down of how I clone using the TOPO TA subcloning kits. The focus will be on the addition of 3’A overhangs.

Insert Preparation

* Setup PCR reactions to amplify your insert. Use a proofreading DNA polymerase.

* Run your DNA gels and cut out your insert. If you do not want any possibility of point mutations or DNA breakage, try excising your bands without exposure to any UV.

* Gel purify your gel cut-outs. I recommend using a kit such as Qiagen's QIAquick Gel Extraction Kit. Elute/resuspend the DNA in nuclease-free water.

Adding 3’A Overhangs

Proofreading DNA polymerases have 5' to 3' polymerization and exonuclease activity as well as 3’ to 5’ exonuclease activity (proofreading). It is the 3’ to 5’ exonuclease activity of a proofreading DNA polymerase which enables it to remove any base-pair mismatch, including any overhanging bases, thereby generating blunt-end PCR products. In contrast, Taq DNA polymerases lack the 3' to 5' exonuclease activity, so while Taq enzymes are not suitable for generating inserts for cloning, they are useful for TA cloning for the addition of 3’A overhangs.

* You will need a Taq DNA polymerase which does not have 3’ to 5’ exonuclease activity. Check the product information sheet. An example of such a Taq is Thermo Scientific's Red Hot Taq DNA Polymerase.

* Using the Red Hot Taq as an example, set up the following:

For x1 reaction:

10x PCR Buffer à 2.5ul

MgCl₂ à 2ul

dATP (10mM stock) à 0.5ul

Red Hot Taq à 0.1ul

Insert DNA (from gel extraction) à 19.9ul

Incubate in a PCR thermal cycler à 72 degrees for 30 minutes. Do not cycle. Cool on ice or 4 degrees when complete.

DNA Precipitation

Cool your reaction on ice and proceed to precipitate your DNA inserts.

* Take the above 25ul reaction and add 2.5ul (which is 1/10^th volume) of 3M pH5.2 NaAc (sodium  acetate). Tap or gently vortex to mix.

* Add 62.5ul (which is 2.5 volumes of ice cold absolute ethanol). Tap or gently vortex to mix.

* Incubate the entire reaction on ice for 30 minutes.

* Centrifuge at 16200xg for 20 minutes.

* Aspirate the supernatant and wash the pellet with 500ul of 70% ethanol.

* Centrifuge at 16200xg for 5 minutes.

* Aspirate the supernatant, air dry the pellet and resuspend in 10ul nuclease-free water.

* Use 4ul for TOPO cloning reaction.