Site-directed mutagenesis
Protocol
1. Vector information: Clontech Cat. #632312 pGFPuv vector
2. Primer information
Name
Sequence
AT64_F | CCA ACA CTT GTC ACT TTC TCT TAT GGT GTT CAA TGC
AT64_R_ | GCATTG AAC ACC ATA AGA GAA AGT GAC AAG TGT TGG
Y66H_F | CCA ACA CTT GTC ACT ACT TTC TCT CAT GGT GTT CAA TGC
Y66H_R_ | GCA TTG AAC ACC ATG AGA GAA AGT AGT GAC AAG TGT TGG
Y145F_F | GGA CAC AAA CTC GAG TAC AAC TIT AAC TCA CAC AAT GTA TAC ATC
Y145F_R | GAT GTA TAC ATT GTG TGA GTT AAA GTT GTA CTC GAG TTT GTG TCC
3. Site-directed mutagenesis
(1) AT64 > observation
(2)
Y66H > observation > Y145F
Day 1. Plasmid preparation / Gel Electrophoresis & mutagenic PCR
1. Reagents and Media
(1) Luria broth agar (LB agar): For growing E.coli
Tryptone
4g
Yeast extract
29
NaCl
49
Agar
6g
Distilled water
400 mL
Prepare 1 bottle for 2 students and autoclave.
* Ampicillin: Add ampicillin when making agar plate, final concentration 50 mg/mL
2. Plasmid preparation (Intron DNA-spin plasmid DNA purification kit)
(1) Centrifuge bacterial culture at 13,000 rpm for 1 min and discard supernatant.
(2) Resuspend cell pellet in 250 uL Resuspension Buffer and vortex.
(3) Add_250 uL Lysis buffer to resuspend cells and mix by inverting the tube 10 times and incubate
for 3 min at RT. * DO NOT VORTEX.
(4) Add 350 wl Neutralization buffer and gently mix by inverting the tube 10 times and incubate
the tube in ice for 5 min.
(5) Centrifuge at 13,000 rpm for 10 min. While waiting for the centrifugation, insert a column into
collection tube.
(6) Transfer supernatant into the column carefully.
(7) Centrifuge at 13,000 rpm for 1 min.
Remove
the column from the collection tube, discard
filtrate in collection tube. And the place the spin column back in the same collection tube.
(8) Add 500 ul Washing buffer A and centrifuge at 13,000 rpm for 1 min. Remove the column
from the collection tube, discard filtrate in collection tube. And the place the spin column
back in
the same collection tube.
(9) Add 700 yL Washing
buffer B and centrifuge at 13,000 rpm for 1 min. Discard filtrate in the
collection tube and place the spin column back in the same collection tube.
(10) Centrifuge at 13,000 rpm for 1 min to dry the filter membrane.
(11) Place the spin column into a clean, new tube. Add 50 uL Elution buffer to the center of the
spin column and wait for 1 min. Centrifuge the tube assembly at 13,000 rpm for 1 min.
3. Mutagenic PCR reaction
10X nPfu enzyme buffer
2 ul
nPfu enzyme DNA polymerase(5U)
1 pl
dNTP mixture
2 pL
Plasmid DNA
1 pl
Forward Primer (10 pmole/yL)
1 pl
Reverse Primer (10 pmole/uL)
1 pl
H20
12 uL
Total
20 pL
Day 2. Dpn | digestion and transformation
1. Dpn I digestion
Prepare the following reaction and incubate the tube at 37°C
Dpnl
0.5 pL (10U)
10X Dpn | buffer
2uL
PCR product
17.5 uh
Total
for 1 hr and 30min.
20 uL
2. Transformation
(1) Thaw cells on ice and add/5} 10 ul of ligation mix to thawed cells, mix gently, and incubate on
ice for
minutes. While you're waiting, pre-heat a hot block (with added water) to 42°C.
(2) Remove the tubes from the ice and immediately put them in the 42°C hot block for 90
seconds. Return the tubes to ice and incubate for 5 min.
(3) Add 0.5 ml of 6
eam
to the cells, transfer to a sterile falcon tube, and incubate at 37°C
(with shaking) for 1 hour.
(4) Plate 100 L of the culture on plates containing the appropriate antibiotic(ampicillin).
(5) Incubate overnight at 37°C. The next morning, pick resultant colonies and make a plasmid
prep (for PCR and restriction enzyme confirmation that the strain has picked up the correct
plasmid).
Day 3. Plasmid preparation / Gel Electrophoresis & mutagenic PCR
1. Plasmid preparation (Intron DNA-spin plasmid DNA purification kit) : Same as above
2. Mutagenic PCR reaction
10X nPfu enzyme buffer
2 pL
nPfu enzyme DNA polymerase(5U)
Tul
dNTP mixture
2 pL
Plasmid DNA
1 pL
Forward Primer (10 pmole/uL)
1 pl
Reverse Primer (10 pmole/pL)
1 pL
H20
12 pL
Total
20 pL
Day 4. Dpn I digestion and transformation / Phenotype observation
1. Dpn I digestion
Prepare the following reaction and incubate the tube at 37°C for 1 hr and 30min.
Dpn I
1 pL (10U)
10X Dpn | buffer
2 yl
PCR product
17 uh
Total
20 uL
2. Transformation
(1) Thaw cells on ice and add 5-10 ul of ligation mix to thawed cells, mix gently, and incubate on
ice for 30 minutes. While you're waiting, pre-heat a hot block (with added water) to 42°C.
(2) Remove the tubes from the ice and immediately put them in the 42°C hot block for 90
seconds. Return the tubes to ice and incubate for 5 min.
(3) Add 0.5 ml of LB medium to the cells, transfer to a sterile falcon tube, and incubate at 37°C
(with shaking) for 1 hour.
(4) Plate 100 pL of the culture on plates containing the appropriate antibiotics(ampicillin).
(5) Incubate overnight at 37°C. The next morning, pick resultant colonies and make a plasmid
prep (for PCR and restriction enzyme confirmation that the strain has picked up the correct
plasmid).
(6) Observe the color change of E. coli on the UV transilluminator.