I've been trying to double mutate (2 base pair mutation to insert ECOR1 site on my plasmid) on my template DNA using quikchange, I've designed my primers and they have 39 % GC content. The protocol I've been using is as follows for a 25μl reaction volume:

Primer F ACA-TCA-TGT-CTT-ATA-CGA-ATT-CAT-CTT-GGA-AAG-TCC-TCT-CCAC

19.4 n.moles ,I added 194 ul ddH2O,then did 1:10 dilution 90 ul ddH2O and 10 ul stock

primer R GTG-GAG-AGG-ACT-TTC-CAA-GAT-GAA-TTC-GTA-TAA-GAA-GAC-ATG-ATGT

12.5 n.moles ,I added 125 ul ddH2O,then did 1:10 dilution 90 ul ddH2O and 10 ul stock

PCR reaction

2.5 μl DMSO (5% final conc)

2.5 μl 10X Pfu Reaction buffer

0.5 μl 10μM forward primer

0.5 μl 10μM reverse primer

4 ul of 10 ng/μl template DNA

1μl 10mM dNTPs

12 μl dddH2O

Add 0.5 μl Pfu turbo polymerase

PCR cycle

95°C for 1 minute (*1)

95°C for 50 second

58°C for 50 second

68°C for 1min/kb (6.34 minutes)

18 cycles

68 degree 6.5 minutes

hold at 4 degree

added 0.5 ul DPN1 and

Incubated for 1 hour at 37°C

transformed into XL10 gold ultra competent cells and got lots of colonies and when i try to digest my sample with ECOR1 it doesn't cut , failed to mutate site

any ideas would be appreciated.