Mutagenesis

QuikChange Lightning Multi Site-Directed Mutagenesis Kit® from Agilent Technologies

Mutant Strand Synthesis Reaction (Thermal Cycling)

1) Prepare the ds-DNA template either by standard miniprep protocols (e.g. StrataPrep Plasmid Miniprep Kit, Stratagene catalog #400761) or by cesium chloride gradient purification.

2) Prepare the mutant strand synthesis reactions for thermal cycling as indicated below. Add the components in the order listed then mix gently by pipetting or tapping the reaction tube.

Reaction Mix:

Reaction Component   	Templates ≤5/>5 kb   	     Control Template   

------------------------------------------------------------------------------
10× QuikChange          2.5 μl   		       2.5 μl
Lightning Multi 
reaction buffer

double-distilled        X μl to final    	       18.5 μl
H20                     volume of 25 μl

QuikSolution   		-/                             -
   			0–0.75 μl           
                        (titrate for eachtemplate)

ds-DNA template   	X μl (50 ng)/                  1 μl QuikChange Multi
   			X μl (100 ng)		       control template

mutagenic primers   	X μl (100 ng each primer       1 μl QuikChange Multi
   			for 1–3 primers; 50 ng         control primer mix
   			each primer for 4–5   	 
                        primers)

dNTP mix   		1 μl   			       1 μl

QuikChange Lightning    1 μl   			       1 μl
Multi enzyme blend

------------------------------------------------------------------------------       		    		 

3) If the thermal cycler to be used does not have a hot-top assembly, overlay each reaction with ~30 μl of mineral oil.

4) Cycle the reactions using the cycling parameters outlined in the table below. (For the control reaction, use a 2.5-minute extension time.)

PCR program:

95°C for 2 minutes
 
30 cycles:
95°C for 20 seconds
55°C for 30 seconds
65°C 30 for seconds/kb of plasmid length
65°C for 5 minutes

4°C on hold

5) Following temperature cycling, place the reaction on ice for 2 minutes to cool the reaction to ≤37°C.

Dpn I Digestion of the Amplification Products

1) Add 1 μl of Dpn I restriction enzyme 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, then immediately incubate each reaction at 37°C for 5 minutes to digest the parental (nonmutated) ds-DNA.