Team:USTC-China/protocols

Gene clone

Gel Extraction

Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)

Protocol

1. Excise gel slice containing DNA fragment of interest.
2. Add 3×sample volume of Buffer DE-A.
Incubate at 75° C for 15-20 min or until gel melts completely.
Add 0.5 × Buffer DE-A volume of Buffer DE-B.

3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)

4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
Repeat wash with Buffer W2
Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.

5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)

DNA digestion

Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.

Single Digestion

	Volume (μl)
Plasmid DNA	Up to 1μg
10×FastDigest Buffer	2
Enzyme	1
ddH2O	To 20
Total	20

2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 30 min.
4. Inactivate the enzyme by adding glycerol DNA loading buffer, or by heating for 5 min at 80°C(only for EcoRI) or 20min at 65°C(only for XbaI)

Double Digestion

	Volume (μl)
Plasmid DNA	Up to 1μg
10×FastDigest Buffer	2
Enzyme1	1
Enzyme2	1
ddH2O	To 20
Total	20

2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 30 min.
4. Inactivate the enzyme by adding glycerol DNA loading buffer, or by heating for 5 min at 80°C(only for EcoRI) or 20min at 65°C(only for XbaI)

Colony PCR

Performed with Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl

	Volume (μl)
10×Taq Buffer	5
dNTP Mix, 2 mM each	5 (0.2 mM of each)
Forward primer	0.1-1.0 μM
Reverse primer	0.1-1.0 μM
25 mM MgCl2*	1-4 mM
Template DNA	Picked from the colony after transformation
Taq DNA Polymerase	0.25 U
ddH2O	To 10
Total	10

*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:

Final concentration of MgCl2, mM	1	1.5	2	2.5	3	4
Volume of 25 mM MgCl2 to be added for 10 μl reaction, μl	0.4	0.6	0.8	1.0	1.2	1.6

3. Gently vortex the samples and spin down.
4. Perform PCR using recommended thermal cycling conditions:

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	95	10 min	1
Denaturation	95	30 s	25~40
Annealing	Tm-5	30 s
Extension	72	1 min/kb
Final Extension	72	5-15 min	1

PCR

Performed with TaKaRa PrimeSTAR® GXL DNA Polymerase

	Volume (μl)
5×PrimeSTAR GXL Buffer	10
dNTP Mixture（2.5 mM each）	4
Forward primer	10~15 pmol
Reverse primer	10~15 pmol
Template DNA	10pg~10ng
PrimeSTAR GXL DNA Polymerase	1
ddH2O	To 50
Total	50

3. Gently vortex the samples and spin down.
4. Perform PCR using recommended thermal cycling conditions:

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	98	8 min	1
Denaturation	98	10 s	25~40
Annealing	55(if Tm<60)	15 s
	60(if Tm>60)
Extension	68	1 min/kb
Final Extension	68	5-15 min	1

Ligation

Performed with TaKaRa Ligation Kit (Solution I)

1. System

	Volume (μl)
Solution I	5
DNA (fragment + carrier)	5

Fragment : carrier (radio in mol)= 3:1~10:1
carrier≥0.03 pmol
2. Gently vortex the samples and spin down.
3. Incubate at 16°C in a low temperature water thermostat for at least 3h.

Plasmid mini-prep

Performed with Shanghai Sangon Plasmid miniprep kit.

1. Preparation
1) Make sure the RNase A has been added into Buffer P1;
2) Make sure the ethanol has been added into Wash Solution
3) Make sure there is no deposit in Buffer P2 and P3

2. Collect the deposit of bacteria from 1.5ml~5ml bacteria liquid (centrifuge at 8,000×g for 2 min)

3. Add 250μl Buffer P1 into the deposit and suspend it thoroughly
4. Add 250μl Buffer P2, mix gently and keep standing in room temperature for 2~4min

5. Add 350μl Buffer P3 and mix gently for 5~10 times
6. Precipitate the bacteria fragments (centrifuge at 12,000×g for 5~10 min) and extract the supernatant (centrifuge at 8,000×g for 30s)

7. Add 500 μl Buffer DW1, centrifuge at 9,000×g for 30s
8. Add 500 μl Wash Solution, centrifuge at 9,000×g for 30s. Repeat this process for one more time. Centrifuge the empty bottle at 9,000×g for 60s.

9. Add 50~100μl Elution Buffer and collect the plasmid.

DNA digestion

1. Remove competent cells (200μl/tube) from freezer and allow to thaw on ice for 3 min
2. Add 3-5 μl of DNA to the cells
3. Incubate on ice for 30 min
4. Heat shock the cells at 42°C for 45 seconds
5. Add 800 μl of LB Broth, and incubate in a shaker at 37°C for 45~60min
6. Centrifuge at 12,000 for 1 min, discard 800 μl supernatant.
7. Re-suspend the pellet in the 200 μl of supernatant and spread onto one agar plate.
8. Incubate the plates overnight at 37°C.

Medium

LB media

Liquid media：

	M/V	1L LB medium
Typtone	1%	10g
Yeast Extract	0.5%	5g
NaCl	1%	10g

Solid media (on the basis of the liquid media)

Agar A

1.5%

15g

M9 media

for 1L 1×media
1 × M9 salt:
Na22HSO4 33.9g/L
KaH2SO415g/L
NaCl 2.5g/L
NH4Cl 5g/L
1. Dissolve 11.3 g Bacto M9 minimal salts in 970mL water.
2. Autoclave to sterilize. 121°C for 20 minutes.

8 mL 50% glycerol
1. Add 4 mL glyerol to 4 mL of H4O
2. Filter sterilize.

2ml 1mol/L MgSO4
1.Dissolve 24.65g MgSO44.7H2O in 100ml water.
2. Autoclave to sterilize. 121°C for 20 minutes.

0.1ml 1mol/L CaCl2
1.Dissolve 1.11g CaCl2 in 10ml water.
2. Autoclave to sterilize. 121°C for 20 minutes.

2g Casamino acids
Dissolve in 1 X M9 salt directly

20ml thiamine
1. Dissolve 0.337 g Casamino acids in 20mL water;
2. Autoclave to sterilize. 121°C for 20 minutes.
Combine above solutions using sterile technique and store at 4°C.

Measurement

Constitutive promoter measurements

1. Streak a LB plate of the strain.
2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic with single colony from the plate.
3. Cultures were grown in test tubes for 16hrs at 37℃ with shaking at 200rpm.
4. Cultures were diluted 1:100 into 3ml fresh medium and grown for 3hrs.
5. Measure the fluorescence(仪器，200ul, excitation, emission, slit width) and absorbance (HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell,path length 10mm,600nm,1.5 nm slit width) every 30 minutes in the next 4hrs.

plac promoter response to IPTG or Glucose

1. Streak a LB plate of the strain.
2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic with single colony from the plate.
3. Cultures were grown in test tubes for 16hrs at 37℃ with shaking at 200rpm.
4. Cultures were diluted 1:1,000 into 3ml fresh medium and grown for 4.5hrs.
5. Stock concentration of the IPTG or glucose (the concentration of the both stock solution is 0.1 M) is diluted and added to different tubes to yield different final concentrations.To ensure the same response time , the IPTG or glucose should be added with a time interval of 2mins between tubes, so do the measurements procedure.
6. Measure the fluorescence(仪器，200ul, excitation, emission, slit width) and absorbance (HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell,path length 10mm,600nm,1.5 nm slit width) every 30 minutes in the next 4hrs.

Phages

Constitutive promoter measurements

1. Inoculate 4ml LB liquid medium in a 30ml test tube with a single colony picked up from the agar slant of E.coli K12 F gal+ (the double lysogen which has both the genome of the lambda phage and the genome of the defective mutant of lambda phage(λdg)). Incubate the colony on a shaking table at 37℃ for 16~20h.
2. Inoculate 4ml LB liquid medium in a 30ml test tube with 0.5ml overnight incubated culture mentioned above and incubate it on a shaking table at 37℃ for 4~6h.
3. Collect the bacteria (centrifuge at 8,000×g for 2 min) and use 4ml of phosphate buffer to resuspend the bacteria deposit.
4. Preheat the UV lamp (30W, whose vertical irradiation distance is 30 cm) for 20min.
5. Add 2ml incubated culture into a small petri dish (diameter=6cm). Put the petri dish onto a magnetic stirrer under the preheated UV lamp. Irradiate for 1 min, open the cap of the petri dish, turn on the magnetic stirrer. After irradiating the dish for accurately 13s, turn off the UV lamp and the magnetic stirrer. Add 2ml double LB liquid media and incubate it without being exposed to light at 37℃ for 2h.
6. Add 4ml incubated culture mentioned above and 0.2ml chloroform into sterilized centrifuge tube. Shake violently for 30s and keep standing for 5min. Centrifuge at 3000 r/min for 10 min.
7. Transfer the supernatant into a test tube, add sterilized glycerol to 30% (V/V) and store the lysate at 4℃.

Lambda phage infecting assay

1. Inoculate 4ml LB liquid medium in a 30ml test tube with a strain you want to test. Incubate the bacteria on a shaking table at 37℃ for 16~20h.
2. Inoculate 4ml LB liquid medium in a 30ml test tube with 0.5ml overnight incubated bacteria mentioned above and incubate it on a shaking table at 37℃ for 4~6h.
3. Prepare the sterilized LB solid media (in which the agar A is only 0.7% (W/V)) and keep it as liquid in a water thermostat at 48℃. Add 0.5ml incubated bacteria mentioned above and lambda phage lysate (the volume is depend on the concentration you want to dilute to). Mix the media thoroughly and pour it onto a standard LB solid media (in which the agar A is 1.5% (W/V)). Be careful to make the newly poured layer of media as smooth as possible.
4. Incubate the media at 37℃ for 12~16h after the upper layer of media is solidified. Observe the media and count the number of plaque.