Kit-Free Site Directed Mutagenesis Protocol

Molecular biologists are familiar with the QuikChange® Site-Directed Mutagenesis Kit that allows rapid intoduction of a point mutant into a plasmid/vector/mammalian expression construct. Briefly, the protocol first involves a thermocycling/PCR step with mutagenic primers, followed by a DpnI digestion step to digest the methylated parental/wild-type plasmid, and finally transformation into competent cells for nick repair.

 An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

 

Most experts we’ve talked to still use this technique, but don’t see the point of an expensive kit. Instead they use their own protocols with inexpensive enzymes and reagents bought separately. One such protocol involves the following:

“QuickChange” Protocol for Site-Directed Mutagenesis

Step 1: Design mutagenesis oligos (Primer 1 and Primer 2) using the website PrimerGenesis

Step 2: Order high quality custom oligos from tebu-bio

Step 3: Set up this reaction:

Plasmid DNA (50ng/ul) 1  ul
10x Reaction Buffer (comes with polymerase) 5 ul
50 mM MgCl2 (comes with polymerase) 1 ul
dNTPs (10mM) 1 ul
DMSO 2.5 ul
Primer 1 (10uM) 1 ul
Primer 2 (10uM) 1 ul
AccuStar™ DNA polymerase* 1 ul
Water 36.5 ul
TOTAL 50 ul

* Due to inherent 3’-to-5’ exonuclease activity of this very high fidelity enzyme, the polymerase must be added last to the reaction in order to prevent primer damage.

Step 4: Run the following PCR Cycle in a thermocycler

95°C 5 min; 15 cycles of (95°C 30sec, 60°C 1 min, 68°C 15 min*), 68°C 10 min, 10°C forever.

*for large (>9kb) plasmids, increase the extension time to 20min.

Note: run a negative control reaction without polymerase enzyme.

Step 5: Add 1 ul of Dpn1 enzyme to PCR tubes and leave at 37C for 2 hours

Step 6: Prepare a 1% agarose gel with GreenView™ Plus DNA Gel Stain

Step 7: Load 10ul of completed reaction mixed with SafeGreen™ Loading Dye next to 1kb DNA ladder and run the gel at 150V for 10 min to determine if PCR was successful. You should not see a product for the ‘negative control’ tube that had no polymerase. Troublshooting: If PCR reaction yields no product, then do gradient for the annealing temperature. The DMSO is extremely important, so keep this in the reactions.

Step 8: Mix PCR reaction with 500ul of PB buffer and purify on column. Wash with PE buffer and elute with 50 ul of water. Use 10ul of for transformation of TransforMax™ EC100™ competent cells.

 

Interested in using this protocol to your lab? Here is a list of products you will need:

Item Name Catalog Number
2′-dNTP Set 040N-2505-25 
Accustar DNA Polymerase 218ME-0067-05 
Molecular Biology Grade Agarose 218EP-0010-05 
GreenView Plus DNA Gel Stain 217N101 
SafeGreen Loading Dye 217D012 
TransforMax EC100 Chemically Competent E. coli 035CC02810 
1kb DNA Ladder 217D001

 

 

 

Legal Notes: QuikChange® is a registered trademark of Stratagene.

5 tips for working with unstable ORF expression clones

Mammalian expression constructs for certain genes such as human c-KIT are notorious for undergoing frequent recombinations during cloning and transformation steps in molecular biology labs. Experts suggest that certain genes are “toxic” to bacteria thus leading to a situation in which recombined plasmids are favored. While the molecular mechanisms for this toxicity may be unclear, the end result is that efforts to amplify, subclone, mutate, or make derivative vectors often result in a new plasmid with unwanted sequence errors.

Online science discussion forums such as ResearchGate include a variety of strategies proposed by researchers experiencing this kind of plasmid instability. Suggestions include advice such as culturing the bacteria at room temperature rather than 37°C, culturing on plates rather than in flasks, using low copy number EPI400 competent cells, picking the small colonies rather then the large ones from the LB plate, replacing the ampicillin resistance cassette to prevent satellite colonies (so you can see the small colonies), or using a Gateway vector.

But which methods work best?

[Read more…]

Alternatives to the “mega-brands” of molecular biology

If you don’t buy luxury brand clothes, why are you still buying luxury brand molecular biology kits?

Let me guess… you might use those blue and red boxed Qiagen® kits for DNA and RNA purification, maybe TRIzol® if you’re a real rebel. You probably also use a SuperScript® RT Kit for reverse transcription and you can’t keep track of which company most recently bought Invitrogen® to control that logo that you’re loyal to.

Maybe it used to be the case that these were the only companies making these kits, so you really had no choice. Now, many of these products are essentially commodities, but you’re still used to purchasing the “first to market” brand because it works and it would probably cause trouble for your lab to change brands. The perceived “switching costs” are large part of the reason for this persistent brand loyalty, and part of the reason why ThermoFisher was willing to pay $13.6 billion for the Life Technologies brand in 2013. There is an entire arm of corporate finance devoted to assigning the value to a brand. Essentially, it tries to estimate how much more customers are willing to pay for a commodity item just because of the company logo on the side of the package.

Here, we examine some alternatives to 2 of the biggest molecular biology mega-brands that seem to be able to keep their loyal customers by placing their familiar brand name on a relatively low value item:

1 – Alternatives to Lipofectamine® Reagents

There’s the one with the blue cap, the one with the red cap, and the one with the green cap, and most people don’t really understand the difference, but cell biologists seem to love these transfection reagents and are willing to pay a heavy premium for them.
Compared to the prices announced in 2015 on the Life Tech website for the 1.5 mL size Lipofectamine® 2000, similar products from competitors are available for about 1/4 the price:

LipoD293 DNA (Ver. II)  1 ml

– Enhanced conjugate with liposome for maximal efficiency
– Best for lentivirus, antibody and protein production

GenMute™ siRNA  1 ml

– Exceptionally good for siRNA transfection
– Excellent for DNA/siRNA co-transfection

LipoJet™ DNA  1 ml

– Fluorinated cationic lipids
– Best transfection efficiency with least toxicity

Validation data for Lipojet™, for example, suggest that it is superior to many other commercial transfection reagents including “L2K”::

Lipojet Multiple Cell Line Data

Lipojet Multiple Cell Line Data

LipoJet_L2K_PolyJet_293T

Given the vast selection of transfection reagents available today, the best option is usually to contact a technical specialist (at tebu-bio for example) for advice on the most suitable ones for your research.

2 – Alternatives to Qiagen® and TRIzol®

Whether you’re using the DNeasy® and RNeasy® kits or TRIzol® products for DNA and RNA extraction, chances are you just keep buying the same kit you learned to use when you were a student. Sure, there are companies making less expensive column-based kits, and there are even protocols online explaining how to regenerate the used columns and make your own buffers. You can also find generic RNAzol RT RNA Isolation Reagent. 

The advances we really like, however, are the purification systems that work without columns and without dangerous solvents. Here are a few of our favourites:

QuickExtract™ DNA Extraction Solution The fastest, least expensive option when you just need to purify DNA for PCR.

QuickExtract™ RNA Extraction Kit  Available in two sizes: the extremely competitively priced 5mL kit contains sufficient material to perform 50 RNA extractions, while the 50mL kit contains sufficient material to perform 500 RNA extractions.

MasterPure™ DNA and RNA Purification Kits  These are for the times when you need the highest quality nucleic acid for next generation sequencing, qPCR, or PCR. Produced by the RNA biology experts at Epicentre (an Illumina company), MasterPure™ Kits are available for nearly every application and source including mammalian cells, yeast, blood, Gram Positive bacteria, and Plant Leaves.

As is the case with transfection reagents, choosing a new nucleic acid purification kit is not always easy, so feel free to contact a technical specialist at tebu-bio for advice.

To get in touch, just leave your comments or questions below.

 

DNeasy® and RNeasy®, and Qiagen® are registered trademarks of the Qiagen group. TRIzol® is a registered trademark of Molecular Research Center, Inc. Invitrogen® and SuperScript® are registered trademarks of Invitrogen. Lipofectamine® is a registered trademark of Life Technologies, Inc.