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.

tebu-bio now distributes Epicentre-Illumina throughout Europe

tebu-bio’s 9 local offices throughout Europe have been bringing innovations to life science researchers for decades. For the past 8+ years, the company’s headquarters in France has served as the exclusive French distributor for Epicentre® Biotechnologies, a US-based company well-known for its unique enzymes and technologies for RNA biology work and Next Generation Sequencing Library prep. As of January 1, 2015, tebu-bio will be distributing Epicentre® products throughout Europe*.

tebu-bio is actively present throughout Europeepicentre_biotechnologies-tebu-bio-20120808162728

Among the most innovative Epicentre® products are those used for so called “tagmentation”, a process that allows researchers to produce libraries for DNASeq and RNASeq much more quickly. Indeed, Illumina®’s acquistion of Epicentre® and the company’s Nextera® DNASeq library prep kits was undoubtedly motivated by this recognized advantage. Similarly Epicentre®’s ScriptSeq Kits have reduced the time required to prepare RNASeq libraries from days to hours using a patented terminal tagging process. While kits and Ribo-Zero™ brand rRNA removal kits are extremely popular with sequencing platforms, RNA biologists and scientists wishing to customize their library prep rely heavily on Epicentre for their innovative enzymes.

For example, groups have published how one can use Epicentre®’s Ez-Tn5™ transposase enzyme to produce high quality DNASeq libraries. (see: doi:10.1101/gr.177881.114). Similarly, this enzyme is useful for tagmentation-based whole genome bisulfite sequencing for epigenetic studies, particularly when very little DNA is available (see: doi:10.1038/nprot.2013.118 and doi:10.1101/gr.136242.111).

Epicentre®’s CircLigase™ II is another favorite of researchers as it is able to attach the 5′ end of a single-stranded DNA molecule to it’s own 3′ end to allow so called rolling-circle replication and transcription for protocols such as iCLIP. Similarly, Epicentre®’s End-It™ DNA Repair and Fast-Link™ DNA Ligation Kits are useful for molecular biology work and library prep as they allow for the blunting and ligation of noncompatible or damaged double stranded DNA overhangs.

Epicentre®’s recent discontinuation of tobacco acid pyrophosphatase (TAP) has served as a reminder of how this company has routinely brought innovative enzymes to the RNA biology community. Among the hidden gems not yet discovered by many researchers are Epicentre®’s MasterPure™ DNA and RNA Purification Kits which function similar to TRIzol® in that they purify DNA or RNA without columns and yet they do not require dangerous organic solvents. Epicentre®’s MonsterScript™ RT Kits that compete with Superscript® III, are also worth trying.

tebu-bio’s distribution of the Epicentre® brand throughout Europe will create some unique bundling opportunities for European customers. Rather than purchasing a single enzyme from each supplier and paying for dry ice shipping multiple times, customers will be able to combine purchases from tebu-bio’s list of hundreds of brands. In particular, many of tebu-bio’s existing Epicentre® customers request combined quotes for enzymes and high quality custom modified oligonucleotides produced by TriLink™ Biotechnologies.

*Update May 6, 2015: tebu-bio’s distribution agreement of Epicentre® (an Illumina company) products includes Austria, Belgium, Bulgaria, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Italy, Latvia, Liechtenstein, Lithuania, Luxembourg, Malta, Monaco, Netherlands, Norway, Republic of Ireland, Republic of Cyprus, Romania, Poland Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland, and the United Kingdom.

Gateway® cloning technology: Is it as easy as they say?

Molecular biology experts have been telling us for a while that the Gateway® cloning vectors are easy-to-use, saving them significant time and effort.

Basically, the technology allows you to start with a single Open Reading Frame (ORF) and pop it into any kind of vector you want without having to think about different restriction sites in the Multiple Cloning Sites in your mammalian, bacterial, lentiviral, etc. expression vectors.

[Read more…]

PrimerGenesis an online tool for today’s molecular biologists

Now that custom gene synthesis costs have made ordering a DNA plasmid with any desired sequence extremely affordable, it is difficult to see the importance of teaching young molecular biologists how to design primers for site directed mutagenesis, adding tags, or C-terminal truncations. I recall during my PhD how our lab’s molecular biology guru tried to teach me how to design mutagenesis oligos to introduce a novel restriction site and how important the GC-clamp was.

[Read more…]