Sense primers used for mutagenesis and restriction enzymes employed for isolating the mutations and replacing the corresponding fragment of the template
Mutant . | Primer . |
|---|---|
| Ala221Val | 5′-ACA GTT TGT GTC CAT GAC CAC ATC AGCG-3′ |
| Ala221Gly | 5′-ACA GTT TGT GGC CAT GAC CAC ATC AGC-3′ |
| Glu275Gln | 5′-ACT GTG GGC CCA CAG GGA AAG TGG-3′ |
| Cys220Ala | 5′-GAT ATA ACA GTT GCT GCC CAT GAC CAC-3′ |
| Cys301Ala | 5′-CAT TGA CAT TAA AAA CGC CCC AAA GAA AAC CAG G-3′ |
Mutant . | Primer . |
|---|---|
| Ala221Val | 5′-ACA GTT TGT GTC CAT GAC CAC ATC AGCG-3′ |
| Ala221Gly | 5′-ACA GTT TGT GGC CAT GAC CAC ATC AGC-3′ |
| Glu275Gln | 5′-ACT GTG GGC CCA CAG GGA AAG TGG-3′ |
| Cys220Ala | 5′-GAT ATA ACA GTT GCT GCC CAT GAC CAC-3′ |
| Cys301Ala | 5′-CAT TGA CAT TAA AAA CGC CCC AAA GAA AAC CAG G-3′ |
Changes introduced are underlined and in bold; antisense counterpart primers are not shown. The size of each cleaved fragment that was isolated and ligated into a template lacking the corresponding fragment is shown in parentheses. Each mutant was cleaved by the enzymes Bln1 and Bsu361, yielding a fragment of 2 kb.