DAPI Staining of Live Yeast Cells
1. Grow yeast cells to mid log phase (OD600-0.5).
2. Centrifuge, 2500 rpm for 1 min, then dump out supernatant.
3. Add fresh medium 200-400 uL.
4. Add DAPI (0.1 mg/mL in stock) to the final concentration of 5 ug/mL.
5. Grow the cells in shaking incubator (30 °C) for 30 min.
6. Centrifuge, 2500 rpm for 1 min, then dump out supernatant.
7. Wash cell pellets with fresh medium twice, centrifuge (2500 rpm) for 1 min each
time.
8. According to cell amount, re-suspend cells in fresh medium.
Enzyme Digestion (eg. Two enzymes)
System (50 uL):
Enzyme I
Enzyme II
10Buffer
BSA
DNA/Plasmid + H2O
2.25 uL
2.25 uL
5 uL
0.5 uL
up to 50 uL
Gel Purification
1. Measure the weight of gel sample.
2. Add 3 volumes of QG Buffer into 1 volume of gel sample.
3. Put the mixture to 55 °C water bath, until gel being melted. (Shake the tube every
few minutes.)
4. Add 1 volume of isopropanol to the tube and mix, then add 750 uL mixture to pink
blue filtration column.
5. Centrifuge, 3000 rpm for 30 sec.
6. Centrifuge, 12,000 rpm for 1 min.
7. Transfer filtrate into upper column again, and centrifuge 12,000 rpm for 1 min.
8. Repeat step 7.
9. Dump out filtrate and add 750 uL PE Buffer into column, centrifuge 12,000 rpm
for 1 min.
10. Dump out filtrate, centrifuge 13,200 rpm for 1 min. Transfer column into the
tube and air-dry.
11. Add 20 uL deionized water/EB Buffer into the tube, and wait for 5 min.
12. Centrifuge, 12,000 rpm for 1 min.
13. Transfer filtrate into column and centrifuge 12,000 rpm for 1 min.
14. Repeat step 13.
15. Throw away column and keep the tube.
Ligation
System (10 uL):
T4 Ligase
10Buffer
Vector DNA
Insert DNA
Ligation into T-vector
System (10 uL):
Solution I
pMD18T
Insert DNA
0.5 uL
1 uL
1 uL
7.5 uL
5 uL
0.5 uL
4.5 uL
PCR
Chromosome PCR
System (50 uL):
Pfx
10Buffer
MgSO4 (50 mM)
dNTP (2.5 mM)
Primer-F (10 uM)
Primer-R (10 uM)
Template
H2O
0.4 uL
7 uL
1 uL
6 uL
1.5 uL
1.5 uL
1-5 uL
up to 50 uL
Colony PCR
System (10 uL/tube):
rTaq
10Buffer
dNTP
Primer-F (100 uM)
Primer-R (100 uM)
H2O
Template (single colony)
0.05 uL
1 uL
0.8 uL
0.05 uL
0.05 uL
up to 10 uL
Extract DNA fragments from PCR products:
1. Add 0.1 volume of NaAc into one volume of PCR products.
2. Add 2 volumes of ethanol into one volume of PCR products.
3. Put the mixture into -20 °C refrigerator, keeping for overnight.
4. Centrifuge and separate out DNA fragments.
5. Use 75% ethanol to wash DNA fragments for three times.
Plasmid Extraction
1. Grow yeast cells to mid log phase (OD600-0.5).
2. Centrifuge, 4000 rpm for 5 min, then dump out supernatant.
3. Add 200 uL P1 Buffer.
4. Add 200 uL P2 Buffer. Invert the tube for 4-6 times softly, and solution turns blue.
5. Add 280 uL N3 Buffer. Invert the tube for 4-6 times immediately, and solution
turns colorless.
6. Centrifuge, 12,000 rpm for 5 min.
7. Transfer supernatant into blue filtration column and mark it.
8. Centrifuge, 3000 rpm for 3 sec, then transfer filtrate into upper column again.
9. Repeat step 8 for 3 times.
10. Dump out filtrate. Add 700 uL PB Buffer; centrifuge 12,000 rpm for 1 min.
11. Add 700 uL PE Buffer; centrifuge 12,000 rpm for 1 min.
12. Repeat step 11.
13. Dump out filtrate and centrifuge 13,200 rpm for 1 min.
14. Transfer upper column into a tube and dry it.
15. Add 80 uL deionized water, centrifuge 12,000 rpm for 1 min.
16. Transfer filtrate into upper column, centrifuge 12,000 rpm for 1 min.
17. Repeat step 16.
18. Throw away column and keep the tube.
Plasmid Transformation
E. coli
1. Add 1 uL plasmid into one tube of competent cells (~100 uL) and mix.
2. Put the tube on ice for 10 min.
3. Put tube in 42 °C water bath for 45 sec.
4. Put tube back on ice for 2 min.
5. Add 800 uL LB Broth into tube and put it in 37 °C shaking incubator, recovering
for 20 min.
6. Centrifuge 3000 rpm for 1 min.
7. Draw out some supernatant and mix the rest medium.
8. Spread plates (with specific antibiotics) before fire.
9. Put plates into 37 °C warm room for 12-16 h.
Yeast
System (360 uL):
PEG (50% w/v)
LiAc (1 M)
ssDNA (10 mg/mL)
Plasmid (200-500 ug)
H2O + Cell
240 uL
36 uL
5 uL
5 uL
74 uL
1. Put the mixture into 30 °C shaking incubator for 20 min.
2. Put the tube into 42 °C water bath for 15-20 min.
3. Use deionized water wash cells for one time.
4. Spread plates before fire.
5. Put plates into 30 °C warm room.
Medium
E. coli
LB Broth (20g/L)
Yeast
1. SD-His medium (glucose)
SD minimal medium (glucose)
-His/-Leu/-Trp DO supplement
D-Leu
D-Trp
H2O
26.7 g/L
0.62 g/L
0.1 g/L
0.05 g/L
up to 1 L
2. SD-His medium (galactose)
Galactose
Yeast Nitrogen base without amino acids
-His/-Leu/-Trp DO supplement
D-Leu
D-Trp
H2O
3. SC medium
SD minimal medium (glucose)
-Leu DO supplement
L-Leucine
20 g/L
6.7 g/L
0.62 g/L
0.1 g/L
0.05 g/L
up to 1 L
26.7 g/L
0.69 g/L
0.1 g/L
H2O
up to 1 L