1 Stock solutions
1.1 Ampicillin: 100 mg/mL.
1 g Amp + 10mL water. Filter sterilize, freeze in aliquots.
1.2 Kanamycin: 50 mg/mL
0.5 g Kan + 10mL water. Filter sterilize, freeze in aliquots.
1.3 Spectinomycin: 10 mg/mL
0.1 g Amp + 10mL water. Filter sterilize, freeze in aliquots.
1.4 Chloramphenicol:
1.5 1M IPTG:
2.4 grams +10mL Water. Filter sterilize, freeze in aliquots.
1.6 50×TAE electrophoresis buffer:
Tris base 242 g
Glacial acetic acid 57.1ml
Na2EDTA·2H2O 37.2 g
H2O to 1 liter
1.7 LB medium
Tryptone 10 g
Yeast Extract 5 g
NaCl 10 g
PH？
Add H2O to 1L
2 Antibiotics
2.1 Ampicillin: 100 μg/mL
2.2 Kanamycin: 50 μg/mL
2.3 Spectinomycin: 100 μg/mL
2.4 Chloramphenicol:
2.5 For co- transformation: Ampicillin 50 μg/mL, Spectinomycin 25 μg/mL, Kanamycin 25 μg/mL
3 Cell transformation
3.1 Remove competent cells on ice to thaw.
3.2 Add 1-10 μl of DNA to the cells.
3.3 Incubate on ice for 20 min.
3.4 Spread onto LB + antibiotic agar plate. Incubate overnight at 37℃.
4 Glycerol stock
4.1 Pick a colony from plate and grow them to OD600=0.6 in LB containing antibiotic.
4.2 150 μl of glycerol add to 850 μl of cell suspension in freezing tube.
4.3 Mix and freeze at -80℃.
5 Miniprep: AxyPrep Plasmid Miniprep Kit. Follow the manufacturer’s protocols.
6 DNA Agarose Gels:
6.1 Weigh out 0.2g of agarose. Add 20mL of 1×TAE.
6.2 Microwave until dissolved.
6.3 cool to about 60℃. Add 2uL of 10,000×Gel Red.
6.4 Pour gel slowly into the tank. Cool in cold room for 0.5 hour.
7 PCR:
7.1 Use TaKaRa Premix Taq Version 2.0. Follow the manufacturer’s protocols.
7.2 Set up the following reaction in a PCR tube on ice.
| Premix Taq |
12.5 μl |
| Template |
0.1 ng - 10 ng |
| Primer F |
0.5 μl |
| Primer R |
0.5 μl |
| Nuclease-free water |
To 25 μl |
| Total volume |
25 μl |
7.3 PCR cycle:
94℃ 3min
30cycles
94℃ 30s
55℃ 30s
72℃ 1min
72℃ 5min
8 PCR Purification: AxyPrep PCR Clearnup Kit. Follow the manufacturer’s protocols.
9 Restriction Digest:
9.1 Instruction
9.2 EroRI
9.3 PstI
9.4 ……
10 Gel Extraction
10.1 Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
10.2 Weigh the Gel.
10.3 Use AxyPrep PCR Clearnup Kit. Follow the manufacturer’s protocols.
11 Ligation:
11.1 Use fermentas T4 DNA Ligase. Follow the manufacturer’s protocols.
11.2 Set up the following reaction in a microcentrifuge tube on ice.
| Linear vector DNA |
20 - 100ng |
| Insert DNA |
10:1 molar ratio over vector |
| 10*T4 DNA Ligase Buffer |
2 μl |
| T4 DNA Ligase |
1 μl |
| Nuclease-free water |
To 20 μl |
| Total volume |
20 μl |
11.3 Incubate 30 min at 22℃.
11.4 Heat inactivation of T4 DNA ligase at 65℃ for 10 min. (not necessary)
11.5 Use 10 μl of the mixture for transformation of 100μl competent cell.
12 Growth Curve:
12.1 Set up cells culture in ~2mL LB + antibiotic.
12.2 Grow at 37℃, 250 rpm overnight.
12.3 Dilute in LB + antibiotic so that final OD600 is ~0.05 for each culture.
12.4 Take OD600 of freshly dilute cultures. Another dilution may be necessary.
12.5 Grow at 37℃, 250 rpm.
12.6 Take OD600 at 30mins.
12.7 Once OD600 has reached ~0.1, begin taking ODs every 20mins.
12.8 When OD600 =0.2， split cultures in half and induce half with 2mM IPTG.
12.9 Once an OD 1.0 is reached dilute the culture 1:1 with media to take an accurate OD. Then multiply the OD value by the dilution factor.
13 Split GFP experiments
13.1 Co-transformation:
13.1.1 Mix the plasmids of D0, FA and FB.
13.1.2 Transform the mixed plasmids to BL21*(DE3)
13.2 Pick a colony from the plate Co-transformation. Grow in LB(50 μg/mL Ampicillin, 25 μg/mL Spectinomycin, 25 μg/mL Kanamycin)
13.3 Induce the cells by 0.2M IPTG for 2 hours at mid-log phase.
13.4 Wash the cells twice with equivalent PBS
13.5 Test with Synergy H1 Hybrid reader
13.6 Take picture with Confocal Scanning Microscope.
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