Editing Quick Change (section)

= Old Protocol: Quick Change<br>  =

== PCR  ==

1. Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence. Purify these oligonucleotide "primers" prior to use in the following steps. 

2. Prepare the control reaction as indicated below: 

5 ÎŒl of 10Ã reaction buffer (see Preparation of Media and Reagents)<br>2 ÎŒl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/ÎŒl)<br>1.25 ÎŒl (125 ng) of oligonucleotide control primer #1 

[34-mer (100 ng/ÎŒl)] 1.25 ÎŒl (125 ng) of oligonucleotide control primer #2<br>[34-mer (100 ng/ÎŒl)] 1 ÎŒl of dNTP mix<br>39.5 ÎŒl of double-distilled water (ddH2O) to a final volume of 50 ÎŒl 

Then add<br>1 ÎŒl of PfuTurbo DNA polymerase (2.5 U/ÎŒl) 

3. Prepare the sample reaction(s) as indicated below:<br>Note Stratagene recommends setting up a series of sample reactions using various concentrations of dsDNA template ranging from 5 to 50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA<br>template) while keeping the primer concentration constant. 

5 ÎŒl of 10Ã reaction buffer<br>X ÎŒl (5â50 ng) of dsDNA template<br>X ÎŒl (125 ng) of oligonucleotide primer #1<br>X ÎŒl (125 ng) of oligonucleotide primer #2<br>1 ÎŒl of dNTP mix 

ddH2O to a final volume of 50 ÎŒl 

Then add<br>1 ÎŒl of PfuTurbo DNA polymerase (2.5 U/ÎŒl) 

4. If the thermal cycler to be used does not have a hot-top assembly, overlay each reaction with ~30 ÎŒl of mineral oil.<br>5. Cycle each reaction using the cycling parameters outlined as follow (For the control reaction, use a 5-minute extension time and run the reaction for 18 cycles.) 

95°C 30 seconds, 1 cycle<br>12â18 cycles of 95 °C 30 seconds, 55 °C 60 seconds, 68°C 1min/kb 

<br> 

6. Adjust segment 2 of the cycling parameters in accordance with the type of mutation desired (see the follow):<br>Point mutations 12<br>Single amino acid changes 16<br>Multiple amino acid deletions or insertions 18 

Note: If desired, amplification may be checked by electrophoresis of 10 ÎŒl of the product on a 1% agarose gel. A band may or may not be visualized at this stage. In either case proceed with Dpn I digestion and transformation 

== Dpn I Digestion of the Amplification Products  ==

Note: It is important to insert the pipet tip below the mineral oil overlay when adding the Dpn I restriction enzyme to the reaction tubes during the digestion step or when transferring the 1 ÎŒl of Dpn I-treated DNA to the transformation reaction. Stratagene suggests using specialized aerosol-resistant pipet tips, which are small and pointed, to facilitate this process. 

1. Add 1 ÎŒl of the Dpn I restriction enzyme (10 U/ÎŒl) directly to each amplification reaction below the mineral oil overlay using a small, pointed pipet tip. 

2. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA. 

== Transformation of XL1-Blue Supercompetent Cells  ==

Notes Please read the Transformation Guidelines before proceeding with the transformation protocol. XL1-Blue cells are resistant to tetracycline. If the mutagenized plasmid contains only the tetR resistance marker, an alternative tetracycline-sensitive strain of competent cells must be used. 

1. Gently thaw the XL1-Blue supercompetent cells on ice. For each control and sample reaction to be transformed, aliquot 50 ÎŒl of the supercompetent cells to a prechilled 14-ml BD Falcon polypropylene round-bottom tube. 

2. Transfer 1 ÎŒl of the Dpn I-treated DNA from each control and sample reaction to separate aliquots of the supercompetent cells.<br>Note Carefully remove any residual mineral oil from the pipet tip before transferring the Dpn I-treated DNA to the transformation reaction. As an optional control, verify the transformation efficiency of the XL1-Blue supercompetent cells by adding 1 ÎŒl of the pUC18 control plasmid (0.1 ng/ÎŒl) to a 50-ÎŒl aliquot of the supercompetent cells. Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes. 

3. Heat pulse the transformation reactions for 45 seconds at 42°C and then place the reactions on ice for 2 minutes.<br>Note This heat pulse has been optimized for transformation in 14-ml BD Falcon polypropylene round-bottom tubes.<br> 

[[Category:Protocols]]