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PCR Random Mutagenesis

 Materials

 1.     Parental plasmid

2.     Oligos surrounding region to be mutagenized

3.     Error-prone PCR buffer (10x is 100mM Tris-HCl, pH8.3; 500mM KCl, 70mM MgCl2, 0.1% (w/v) gelatin.)

4.     10mM MnCl2 (stock)  (make sure there is no brown coloring to stock soln; if there is it means it oxidized to Mn3+ which will kill your polymerase)

5.     dNTPs

6.     Taq polymerase

 

Method

 

Add:    10l of 10X Error-prone PCR buffer,

            10l DMSO (for GC rich template regions)

            3l of 10mM MnCl2

50pmol of each primer,

            10ng of template plasmid,

            dNTPs

                        to 0.2mM of dATP and dGTP;

                        to 1mM of dCTP and dTTP

            5U of Taq polymerase

            H2O to 100l

            -It is preferrable to then split this stock into 10l aliquots and then run these reactions separate and then mix them together after completion of the reaction.

 

PCR:               94° for 30s

                        55° for 1min

                        72° for 1min

                        repeat for 30 cycles

 

Notes:

1)    The PCR cycle temps and times listed above are for PCRing aLP DNA and will probably need to be changed to suit the needs of you and your template.

 

2)    The MnCl2 concentration can be changed to alter the mutagenesis rate according to your wants and needs as an experimental research scientist.  The above protocol calls for 0.3mM MnCl2 which will give an approximate 5 point mutations/kb.

 

3)    The separation of PCR master mix into different aliquots prevents the over-representation of single mutations in your library.  This is because if a mutation occurs in the first rounds of PCR, then this mutation will be present in most or all of the subsequent products, thereby reducing the diversity of your library.

 

4)    Even though PCR mutagenesis is rather robust, there still is an inherent mutagenic bias associated with this method.  There is a PCR mutagenesis kit sold by Stratagene (the GeneMorph system using Mutazyme polymerase) that has an opposite bias.  A combination of these two methods should produce the most diverse library.  (For a very good representation do 15 rounds of this PCR followed by 15 rounds of Mutazyme.)

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