The authors would like to thank University of Southern Denmark open access policy grants.

1. Vector selection
<ol>
	<li>Choose a vector to perform downstream experiments with. Any vector is applicable for this 2- and 3-step PCR method.</li>
	<li>Based on choice of vector, choose appropriate restriction enzymes for cloning.</li>
</ol>
2. Primer design for site directed mutagenesis
<ol>
	<li>Decide between either the 2-step or 3-step PCR strategy (2-step is only for mutations &#60;200 bp from either end of the DNA of interest). For the 2-step PCR, go to step 2.2 and for the 3-step PCR go to step 2.3.</li>
	<li>Design primers for 2-step PCR.
	<ol>
		<li>Design primer 1 and 3 to amplify the DNA of interest and with a 5&#8217; overhang that contains 4 nucleotides (e.g., ATAT or AGCT) followed by the relevant restriction recognition site necessary to clone into the chosen vector.</li>
		<li>Design primer 2 to introduce mutation(s) at the desired site(s) and flank the mutation with 10-15 complementary nucleotides on both sides. Make the primer reverse if the mutation is introduced at the 5&#8217; end or forward if the mutation is introduced at the 3&#8217; end.</li>
	</ol>
	</li>
	<li>Design primers for 3-step PCR.
	<ol>
		<li>Design primer 1 and 4 to amplify the DNA of interest and with a 5&#8217; overhang that contains 4 nucleotides (e.g., ATAT or AGCT) followed by the relevant restriction recognition site necessary to clone into the chosen vector.</li>
		<li>Design primer 2 and 3 to introduce mutation(s) at desired site(s) and flank the mutation by 10-15 complementary nucleotides on both sides. Primer 2 and 3 are reverse complementary.</li>
	</ol>
	</li>
</ol>
3. PCR amplification of wild type DNA for cloning
NOTE: For details on PCR, see6.
<ol>
	<li>Perform PCR6 using primers 1+2 (2-step PCR) or 1+4 (3-step PCR) and use wild type DNA as template to obtain PCR product I. Use the PCR program in Table 1.</li>
	<li>Validate PCR by agarose gel electrophoresis.
	<ol>
		<li>Make an agarose gel solution (2%) by adding 2 g of agarose per 100 mL of 1x Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer. Dissolve the agarose by boiling in a microwave oven. Add the DNA-staining dye ethidium bromide (to a final concentration of ~ 0.5 &#181;g/mL) to the agarose gel solution for visualization.</li>
		<li>Cast agarose gel, place it in an electrophoresis unit, and load PCR samples (mixed with DNA loading dye) and a DNA ladder of known size. Run samples at 75 W for 45 min, or until bands are separated adequately, and visualize bands at an ultra-violet (UV) table or with a gel imaging system.</li>
	</ol>
	</li>
	<li>Purify the PCR product with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
	<li>Store the purified DNA at -20 &#176;C (in Tris-EDTA (TE) buffer &#8211; long term storage) or 4 &#176;C (in dH2O &#8211; short term storage) until used in step 5.</li>
</ol>
4. PCR to introduce site-directed mutations in the DNA
<ol>
	<li>PCR for 2-step PCR (see step 4.2 for 3-step PCR)
	<ol>
		<li>Perform PCR6 using primers 1+2 if mutations are in the 5&#8217; end or 2+3 if the mutations are in the 3&#8217; end and use wild type DNA as a template to obtain PCR product II (see Table 1 for PCR program).</li>
		<li>Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
		<li>Purify the PCR product with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until used in step 5.</li>
		<li>Perform PCR6 using PCR product II as the primer together with primer 3 if mutations are in the 5&#8217; end or primer 1 if the mutations are in the 3&#8217; end and use wild type DNA as template to obtain PCR product III (see Table 1 for PCR program).</li>
		<li>Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
		<li>Purify PCR product with a gel extraction kit (Table of Materials) and measure concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until step 5.</li>
	</ol>
	</li>
	<li>PCR for 3-step PCR
	<ol>
		<li>Perform PCR6 using primers 1+2 and 3+4 in separate reactions and use wild type DNA as template to obtain PCR product II and III (see Table 1 for PCR program).</li>
		<li>Validate PCRs by agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
		<li>Purify PCR products with a gel extraction kit (Table of Materials) and measure the concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O).</li>
		<li>Perform PCR6 using primers 1+4 and use 2-5 ng of both PCR products II and III as the templates (in the same reaction) to obtain PCR product IV (see Table 1 for PCR program).</li>
		<li>Validate PCR by agarose gel electrophoresis as in step 3.2.1 and 3.2.2. 
		NOTE: It is not unusual to get several incorrect bands (can sometimes be reduced by using less template). However, the incorrect PCR products can be ignored if the correctly sized band is excised and gel-extracted.</li>
		<li>If correct, purify PCR product with a gel extraction kit (Table of Materials) and measure concentration of purified DNA with a spectrophotometer (Table of Materials).</li>
		<li>Store purified DNA at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until step 5.</li>
	</ol>
	</li>
</ol>
5. Recombination of wild type and mutant version(s) of DNA into the chosen vector
NOTE: For details on following steps, see7.
<ol>
	<li>Digest purified PCR products I and III (from 2-step PCR) and/or IV (from 3-step PCR) using the relevant restriction enzymes.
	<ol>
		<li>In 20 &#181;L of 1x digestion buffer with 1 &#181;L of each restriction enzyme, digest 200 ng of PCR product at 37 &#176;C for 30-60 min.</li>
	</ol>
	</li>
	<li>Purify digested PCR product by gel-extraction using a gel extraction kit (Table of Materials), measure the DNA concentration of purified DNA with a spectrophotometer (Table of Materials), and store at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until used for step 5.6.</li>
	<li>Digest purified vector with relevant restriction enzymes and treat with alkaline phosphatase to decrease vector re-ligation events. Do not treat PCR products with alkaline phosphatase.
	<ol>
		<li>In 30 &#181;L of 1x digestion buffer with 1 &#181;L of each restriction enzyme and 1 &#181;L of alkaline phosphatase, digest 1,000 ng of the vector at 37 &#176;C for 30-60 min.</li>
	</ol>
	</li>
	<li>Separate digested vector from waste DNA (e.g., uncut vector and cut-out DNA) using agarose gel electrophoresis as in step 3.2.1 and 3.2.2.</li>
	<li>Purify digested vector by gel-extraction using a gel extraction kit (Table of Materials), measure DNA concentration of purified DNA with a spectrophotometer (Table of Materials), and store at -20 &#176;C (in TE buffer) or 4 &#176;C (in dH2O) until used for step 5.6.</li>
	<li>Ligate digested PCR products into digested vector with the reactions specified in Table 2.</li>
	<li>Incubate at room temperature for 2 h or overnight at 16 &#176;C.</li>
	<li>Transform recipient strain (e.g., E. coli K12) with ligation reactions.
	<ol>
		<li>Grow strain to OD600=0.3-0.5 and transfer a 1 mL culture to as many 1.5 mL tubes as ligase reactions.</li>
		<li>Spin at 3,500 x g for 5 min and discard supernatant.</li>
		<li>Resuspend cells in 200 &#956;L of transformation buffer (10 mL of lysogeny broth (LB) with 0.1 g/mL polyethylene glycol 3350, 5% dimethyl sulfate and 20 mM MgCl2).</li>
		<li>Add ligation reaction, and place the tubes on ice for 30 min.</li>
		<li>Heat-shock for 2 min at 42 &#176;C.</li>
		<li>Add 1 mL of LB to the 1.5 mL tubes, and allow phenotypic expression of antibiotic resistance for at least 45 min at 37 &#176;C.</li>
		<li>Spin the cells at 3,500 x g for 5 min, discard 1 mL of supernatant, and resuspend the cells in the remaining supernatant.</li>
		<li>Plate the cells on plates with appropriate antibiotics and incubate overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Identify transformants harboring vectors with successful integration of DNA insert (e.g., by PCR using vector- and insert-specific primers).</li>
	<li>Validate sequence of DNA by sanger sequencing. 
	CAUTION: Do not use the same primers for sequencing as used for step 5.9.</li>
</ol>
6. Using constructed vectors for in vitro and/or in vivo experiments
<ol>
	<li>In vivo experiment 
	NOTE: This is an example of using a vector to express wild type/mutated RNA to characterize post-transcriptional regulation. For further details on western blotting, see8.
	<ol>
		<li>Grow E. coli K12 strains with constructed vectors in appropriate medium and induce expression if required. Harvest samples by centrifugation.</li>
		<li>Prepare samples for sodium dodecyl sulfate&#8211;polyacrylamide gel electrophoresis (SDS-PAGE) by dissolving cell pellets in 1x SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 0.002% bromophenol blue, 5% &#946;-mercaptoethanol, 10% glycerol) and boil at 95 &#176;C for 5 min. 
		NOTE: It is possible to cast a gel or use a commercially available precast gel. The latter was used in the results presented in Figure 3 (Table of Materials).</li>
		<li>Load 107 cells of each sample in separate wells (include a protein ladder), and run gel at 200 V until proteins are separated (approximately 45 min).</li>
		<li>Blot the proteins onto a cellulose-membrane by semi-dry transfer at 80 mA for 1 h.</li>
		<li>Block the membrane with a mixture of proteins (e.g., 5% milk-powder dissolved in 1x Tween 20-Tris-buffered saline (TTBS) buffer).</li>
		<li>Add primary antibody (dissolved in 1x TTBS buffer) that target the protein of interest (e.g., GFP-, FLAG-, or HIS-tagged protein) and incubate for 1 h with gentle agitation.</li>
		<li>Wash membrane in 1x TTBS for 10 min to remove unbound antibodies. Repeat twice more.</li>
		<li>Add secondary antibody (dissolved in 1x TTBS buffer) that targets the primary antibodies and allow for detection (e.g., horseradish peroxidase (HRP)-conjugated secondary antibodies. Incubate for 1 h with gentle agitation.</li>
		<li>Wash the membrane in 1x TTBS for 10 min to remove unbound antibodies. Repeat twice more.</li>
		<li>Visualize the membrane with a technique compatible with the secondary antibodies (e.g., by imaging after incubation with a luminol-derived chemiluminescence, if a HRP-conjugated secondary antibody was used).</li>
	</ol>
	</li>
	<li>In vitro experiment 
	NOTE: This is an example of using the vector as a template for in vitro transcription of RNA to characterize RNA-protein interactions. For further details on EMSA, see9.
	<ol>
		<li>Make in vitro transcripts using a T7 in vitro transcription kit (Table of Materials) and vectors from step 5 as templates.</li>
		<li>Separate RNA transcripts by PAGE on a 4.5% 7 M urea denaturing gel, and extract RNA directly from the gel by electro elution with dialysis tubes (Table of Materials).</li>
		<li>Label RNA (e.g., radiolabeling with &#947;-32P-ATP using T4-polynucleotide kinase (Table of Materials) and purify again with columns (Table of Materials). 
		CAUTION: Before working with radioactive material, consult with the local radiation safety officer.</li>
		<li>Mix labelled-RNA with increasing concentrations of protein in separate reactions in a 1x binding buffer (20 mM Tris, pH 8, 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT)). 
		NOTE: In the presented results (Figure 4), 2 nM of radiolabeled csgD mRNA was mixed with a gradient of 0 to 2 &#181;M Hfq protein (monomer concentration).</li>
		<li>Allow protein and RNA to hybridize before loading hybridization mix on a non-denaturing polyacrylamide gel and run the gel for 1.5 h at 200 V.</li>
		<li>Visualize the gel with a technique compatible with the labeling from step 6.1.3. (e.g., by phosphoimaging if radiolabeling was applied).</li>
		<li>Quantify the relative intensity of the shifted bands with an imaging processing program and fit a curve (sigmoidal) to the data by using a graph and data analysis software (Table of Materials). Based on the fitted curve, dissociation constant (Kd) values can be determined automatically with the software.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bouvier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA+binding+to+5'+mRNA+coding+region+inhibits+translational+initiation.">Small RNA binding to 5' mRNA coding region inhibits translational initiation.</a> Molecular Cell. 32 (6), 827-837 (2008).</li><li>Jorgensen, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+regulatory+RNAs+control+the+multi-cellular+adhesive+lifestyle+of+Escherichia+coli.">Small regulatory RNAs control the multi-cellular adhesive lifestyle of Escherichia coli.</a> Molecular Microbioly. 84 (1), 36-50 (2012).</li><li>Antal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bacterial+RNA+regulates+a+putative+ABC+transporter.">A small bacterial RNA regulates a putative ABC transporter.</a> The Journal of Biological Chemistry. 280 (9), 7901-7908 (2005).</li><li>Andreassen, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sRNA-dependent+control+of+curli+biosynthesis+in+Escherichia+coli:+McaS+directs+endonucleolytic+cleavage+of+csgD+mRNA.">sRNA-dependent control of curli biosynthesis in Escherichia coli: McaS directs endonucleolytic cleavage of csgD mRNA.</a> Nucleic Acids Research. , (2018).</li><li>Thomason, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+RNA+that+regulates+motility+and+biofilm+formation+in+response+to+changes+in+nutrient+availability+in+Escherichia+coli.">A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli.</a> Molecular Microbioly. 84 (1), 36-50 (2012).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> Journal of Visualized Experiments. , (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+The+Western+Blot.">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot.</a> Journal of Visualized Experiments. , (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Biochemistry.+Electrophoretic+Mobility+Shift+Assay+(EMSA).">JoVE Science Education Database. Biochemistry. Electrophoretic Mobility Shift Assay (EMSA).</a> Journal of Visualized Experiments. , (2018).</li><li>Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+site-specific+mutagenesis+without+phenotypic+selection.">Rapid and efficient site-specific mutagenesis without phenotypic selection.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (2), 488-492 (1985).</li><li>Laible, M., Boonrod, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homemade+site+directed+mutagenesis+of+whole+plasmids.">Homemade site directed mutagenesis of whole plasmids.</a> Journal of Visualized Experiments. (27), (2009).</li><li>Wells, J. A., Vasser, M., Powers, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cassette+mutagenesis:+an+efficient+method+for+generation+of+multiple+mutations+at+defined+sites.">Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites.</a> Gene. 34 (2-3), 315-323 (1985).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li></ol>

The authors declare no competing interests.

Site-directed mutagenesis has a broad array of different applications, and here, representative results from an in vivo and an in vitro experiment were included as examples of how to make biological conclusions using the technique. Site-directed mutagenesis has for long been the golden standard for RNA interaction studies. The strength of the technique lies in the combination of introducing relevant mutations with downstream assays and experiments (e.g., western blot or EMSA) to draw conclusions about specific DNA sites ...

DNA is often referred to as the blueprint of a living cell since all structures of the cell are encoded in the sequence of its DNA. Accurate replication and DNA repair mechanisms ensure that only very low rates of mutations occur, which is essential for sustaining correct functions of coded genes. Changes of the DNA sequence can affect successive functions at different levels starting with DNA (recognition by transcription factors and restriction enzymes), then RNA (base-pair complementarity and secondary structure alterations) and/or protein (amino acid substitutions, deletions, additions or frame-shifts). While many mutations do not affect gene function significantly, some mutations in the DNA can have huge implications. Thus, site-directed mutagenesis is a valuable tool for studying the importance of specific DNA sites at all levels.
This protocol describes a targeted mutagenesis approach used to introduce specific mutations. The protocol relies on two different PCR strategies: a 2-step or a 3-step PCR. The 2-step PCR is applicable if the desired mutation is close to either the 5&#39; end or the 3&#39; end of the DNA of interest (&#60;200 base pairs (bp) from the end) and the 3-step PCR is applicable in all cases.
In the 2-step PCR approach, 3 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 3, forward and reverse, respectively), and a single primer is designed to incorporate the mutation. The mutation introducing primer (primer 2) should have a reverse orientation if the mutation is close to the 5&#39; end and a forward orientation if the mutation is close to the 3&#39; end. In the first PCR step, primer 1+2 or 2+3 amplifies a small fragment close to the 5&#39; end or 3&#39; end, respectively. The resulting PCR product is then used as a primer in step two with primer 1 or 3, thus resulting in a PCR product with a mutation in the DNA of interest (Figure 1A).
In the 3-step PCR, 4 primers are designed, in which one set of primers is designed to amplify the DNA of interest (primers 1 and 4, forward and reverse, respectively) and one set of primers is designed to incorporate specific mutations with overlapping complementarity (primers 2 and 3, reverse and forward, respectively). In step one and two, primers 1+2 and 3+4 amplify the 5&#39; and 3&#39; end. In step three, the resulting PCR products from step one and two are used as templates and amplified with primers 1+4. Thus, the resulting PCR product is the DNA of interest with the desired mutation (Figure 1B).
While the mutated DNA can be used for any downstream application, this protocol describes how to re-combine the DNA into a cloning vector. The use of cloning vectors has several advantages such as ease of cloning and specific experimental applications depending on features of the vector. This feature is often used for RNA interaction studies. Another technique for RNA interaction studies is structural probing of the RNA in complex with another RNA1,2 or protein3,4. However, structural probing is only performed in vitro whereas site-directed mutagenesis and subsequent cloning allow for interaction studies in vivo.
Site-directed mutagenesis has been extensively used for RNA interaction studies as presented here. However, the key method regarding 2- or 3-step PCR is applicable to any piece of DNA, and thus not only limited to RNA-interaction studies.
To exemplify the technique and its possible uses, characterization of regions important for post-transcriptional regulation of the mRNA, csgD, of Escherichia coli (E. coli) is used. In E. coli, csgD is targeted by the small non-coding RNA, McaS, in cooperation with a protein, Hfq, to repress protein-expression of CsgD2,4,5. The technique is used to introduce mutations to the base-pairing region between csgD and McaS, and to the Hfq binding site of csgD. The obtained DNA is then cloned into a vector suitable for subsequent experiments. Downstream applications of the technique include both in vivo and in vitro experiments. For illustration, example 1 is characterized in vivo using a western blot assay and example 2 is characterized in vitro using an electrophoretic mobility shift assay (EMSA). In both cases, it is illustrated how site-directed mutagenesis can be used in combination with other techniques to make biological conclusions about a gene of interest.

<table>
	<tbody>
		<tr>
			<td>Anti-GroEL antibody produced in rabbit</td>
			<td>Merck</td>
			<td>G6532</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Azure c200</td>
			<td>Azure</td>
			<td>NA</td>
			<td>Gel imaging workstation</td>
		</tr>
		<tr>
			<td>Custom DNA oligo</td>
			<td>Merck</td>
			<td>VC00021</td>
			<td></td>
		</tr>
		<tr>
			<td>DeNovix DS-11</td>
			<td>DeNovix</td>
			<td>NA</td>
			<td>Spectrophotometer for nucleic acid measurements</td>
		</tr>
		<tr>
			<td>DNA Gel Loading Dye (6X)</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1 %</td>
			<td>Carl Roth</td>
			<td>2218.1</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJET Gel Extraction Kit</td>
			<td>Thermo Scientific</td>
			<td>K0691</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler DNA Ladder Mix</td>
			<td>Fermentas</td>
			<td>SM0333</td>
			<td></td>
		</tr>
		<tr>
			<td>Gerard GeBAflex-tube Midi</td>
			<td>Gerard Biotech</td>
			<td>TO12</td>
			<td>Dialysis tubes for electro elution</td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT Cell</td>
			<td>Bio-Rad</td>
			<td>1704406</td>
			<td>Horizontal electrophoresis system</td>
		</tr>
		<tr>
			<td>Monoclonal ANTI-FLAG M2 antibody produced in mouse</td>
			<td>Merck</td>
			<td>F3165</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Mouse Immunoglobulins</td>
			<td>Dako Cytomation</td>
			<td>P0447</td>
			<td>HRP conjucated secondary antibody</td>
		</tr>
		<tr>
			<td>NucleoSpin miRNA</td>
			<td>Macherey Nagel</td>
			<td>740971</td>
			<td>RNA purification</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Thermo Scientific</td>
			<td>NP0323BOX</td>
			<td>Bis-Tris gels for protein separation</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>New England Biolabs</td>
			<td>M0531S</td>
			<td>DNA polymerase</td>
		</tr>
		<tr>
			<td>PowerPac HC High-Current Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645052</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Immunoglobulins</td>
			<td>Dako Cytomation</td>
			<td>P0448</td>
			<td>HRP conjucated secondary antibody</td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaPlot</td>
			<td>Systat Software Inc</td>
			<td>NA</td>
			<td>Graph and data analysis software tool</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td>PCR machine</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE Buffer (Tris-acetate-EDTA) (50X)</td>
			<td>Thermo Scientific</td>
			<td>B49</td>
			<td></td>
		</tr>
	</tbody>
</table>

site-directed mutagenesis for in vitro and in vivo experiments exemplified with rna interactions in escherichia coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

To investigate RNA interactions regarding post-transcriptional regulation of csgD, a double vector setup was chosen: one to express the csgD mRNA and another to express the small non-coding RNA, McaS. csgD was cloned into pBAD33, which is an arabinose inducible medium-copy plasmid with chloramphenicol resistance and McaS was cloned into mini R1 pNDM220, which is an isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) inducible low copy plasmid with ampicillin resis...

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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

site-directed-mutagenesis-for-vitro-vivo-experiments-exemplified-with

Research

JoVE Journal

Biochemistry

19.7K Views.  University of Southern Denmark. Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Video: Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...