Team:ZJU-China/notebook.htm

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.