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From 2013.igem.org

Protocol

Protocol1

PLASMID DNA

COMMPONENT	20µl REACTION
Nuclease-free water	17µl
10XFastDigest Buffer	2µl
DNA	1µl(up to 1ug)
Incubate at 37℃ in a heat block or water 1 hour

PCR product

COMMPONENT	20µl REACTION
Nuclease-free water	17µl
10XFastDigest Buffer	3µl
DNA	10µl(~0.2ug)
Incubate at 37℃ in a heat block or water 1 hour

Double Digest of DNA

• Use 1 ul of each enzyme and scale up the reaction conditions appropriate.
  
• If the enzyme require different reaction temperatures, start with the enzyme that requires a lower temperature, then add the second enzyme and incubate at the high temperature.

DNA ligation with T4 DNA Ligase

COMMPONENT	20µl REACTION
10X T4 DNA Ligase Buffer*	2µl
Vector DNA (3 kb)	50 ng
Insert DNA (1 kb)	50 ng
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 μl
Incubate at 22℃ in a heat block or water 1 hour

PCR Protocol for Phusion

COMMPONENT	20µl REACTION	50µl REACTION
Nuclease-free water	Add to 20µl	Add to 50µl
2x Phusion Master Mix	10µl	25µl
primer Forward(10uM)	0.2µl	0.5µl
primer Reverse(10uM)	0.2µl	0.5µl
template DNA	1 pg–10 ng	1 pg–10 ng
(DMSO, optional)	(0.6 µl)	(1.5 µl)

Step1
98℃	3min	X1 cycle
Step2
98℃	10s	X30 cycles
50℃	45s
72℃	2min
Step3
72℃	10min	X1 cycles
10℃	20min

Colony PCR Take a 20 µl pipet tip and touch a colony very lightly and dip the tip a couple of times into the 15 µl of water. Repeat for each colony.

COMMPONENT	10µl REACTION
Sterile water	Add to 10µl
10x PCR buffer	1µl
Water with colony	1µl
MgCl2 (25mM)	1µl
primer Forward	0.1µM
primer Reverse	0.1µM
dNTPs (10mM)	0.2µl
Taq	(0.1µl)

Step1
94℃	3min	X1 cycle
Step2
94℃	30s	X30 cycles
55℃	30s
72℃	Amplicon specific (~ 1 minute per kb)
Step3
72℃	10min	X1 cycles
10℃	20min

E. coli Calcium Chloride competent cell protocol

• 1. Streak E.coli cells (DH5a, HB101, GM8) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)

• 2. Allow cells to grow at 37℃ overnight

• 3. Place one colony in 10 ml LB media (+antibiotic selection if necessary), grow overnight at 37℃

• 4. Take 2 ml LB media and save for blank. Transfer 5 ml overnight DH5a culture into 500 ml LB media in 3 L flask

• 5. Allow cell to grow at 37oC (250 rpm), until OD600= 0.4 (~2-3 hours)

• 6. Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min

• 7. Centrifuge cells in at 4oC for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room

• 8. Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min

• 9. Centrifuge cells at 4oC for 10 min at 3,000 g

• 10. Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 ml into (1.5 ml) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months

• 11. Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step

• 12. Incubate the mixture on ice for 30 minutes.

• 13. Transfer the reaction to a 42℃ water for 1min.

• 14. Add 0.9 ml of LB culture to each tube and incubate at 37oC for 1 hour in a roller drum (250 rpm) to allow cells to recover and express the antibiotic resistance marker.
  

• 15. Incubate on ice for 2 minutes.
  

• 16. Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4oC.
  

(A) E. coli cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium.

(B) E. coli cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.

• 17. Incubate all plates overnight at 37oC (agar side up).
  

• 18. Count the number of colonies.

Enzyme Activity Assay with Detecting Cl^- Concentration

Reflecting with the substrate, and releasing the Cl^- is the function of the four kinds of enzyme. So in the stable reaction system the concentration of Cl^- will ascend with the beginning of the reaction. The released Cl^- can reaction with Hg^(2+) from Hg(SCN)_2 and release the SCN^-. When adding to the Fe^(3+), they will react together and produce Fe(SCN)_3, a kind of red complex compound， at the 460nm it has absorption peak.

Chemical equation：

Hg(SCN)2 + Cl- ――→HgCl + SCN-

NH4Fe(SO4 )2 + SCN- ――→ Fe(SCN)3 + (NH4 )2SO4

SolutionⅠ

Hg(SCN)2—Alcohol (4g/L) : 0.4g Hg(SCN)2, 100ml Alcohol；

SolutionⅡ

NH4Fe(SO4)2 : 13.4g NH4Fe(SO4 )2, 67.1ml HNO_3, Water(add up to 1L)

Cl- Standard Curve

• 1.Use Tris-SO4(50mM,PH=8.0) dissolves NaCl to 0mM, 1.0mM, 2.0mM, 4.0mM, 5.0mM, 6.0mM, 8.0mM, 10.0mM

• 2.Add NaCl to 0.9ml solutionⅠ mix 1min, add solution Ⅱ 0.1ml to it

• 3.Measure the spectrum absorption at 460nm.

Our standard curve:

Equation y = 0.0071 + 0.31673*x

R-Square 0.99618

Detect the Activity of Enzyme

• 1.Use LB free from NaCl cultivate E.coli(MC1061 in our test) at 37℃ up to OD=0.6~0.8

• 2.Add to inducer(arabinose in our test) and cultivate at 30℃ for 12hours

• 3.Transfer the 20 ml cell to centrifuge bottles(50ml)

• 3.Centrifuge cells for 10 min at 7,000 g

• 4.Pour off media and resuspend cells with 20ml Tris-SO_4(50mM,PH=8.0)

• 5.Repeat 4-5th step twice for wiping off Cl^-

• 6.Repeat the step 4th and pour off the media again, resuspend cells with 4ml Tris-SO_4(50mM,PH=8.0)

• 7.Transfer 1ml of the cell to Ependorff tubes(1.5ml). Centrifuge and pour off the buffer. Cells stored at -80oC can be used for transformation for up to 1 year.

• 8.Put Ependorff tubes in the Bunsen beaker filled with ice. Crushed by sonicator.

A.Crude detection

1.add 900µl solutionⅠ into tubes for preparation.

2.Tris-SO_4(50mM,PH=8.0), substrate and cell disruption to construct ubstrate(5mM) reaction system.

3.When adding to the cell disruption, mix up totally, transfer 250µl reaction liquid to the prepared tube with 900µl solutionⅠ.

4.add solutionⅡ into the tube.

5.test absorbance at 460nm.

6.figure out the Cl^- concentration according to Cl^- standard curve formula.

B. Supernatant and Sediment Activity Detection

1.add 900µl solutionⅠ into tubes for preparation.

2.centrifuge cell disruption at 18,000g for 40min.

3.tranfer the supernatant to Ependorff tubes(1.5ml).

4.Tris-SO_4(50mM,PH=8.0), substrate and supernatant(or sediment) to construct substrate(5mM) reaction system.

5.repeat the step3-6th of Crude detection.

C.Enzyme Activity Assay

1. add 900µl solutionⅠ into tubes for preparation.

2. centrifuge cell disruption at 18,000g for 40min.

3. tranfer the supernatant to Ependorff tubes(1.5ml).

4.assay supernatant protein concentration(Coomassie brilliant blue).

5.Tris-SO_4(50mM,PH=8.0), substrate and supernatant known concentration to construct substrate(5mM) reaction system.

6. repeat the step3-6th of crude detection.

GC analysis.

For GC analysis, we used a FuLi GC,an FID detector. Separation was performed with a AC5 column(30mm*25um*0.25um), isothermic at 100℃ for 8min, increase with 10℃ per min to 170℃15min on 170℃。

The samples were extracted with diethyl ether containing mesitylene or 1-chlorohexane as an internal standard. Prior to analysis by GLC, the samples were dried over a short column containing MgSO4. The diols were analysed after derivatisation to their acetonides. The diethyl ether was vaporized under a stream of nitrogen and the sample was redissolved in 0.5 ml 2,2-dimethoxypropane. The solution was then shaken for 1 h with 200 mg amberlite IR-120 (H+). After addition of 50 mg NaHCO3 the organic phase was analysed by GLC.

Retention times were 6.9 min (TCP), 7.2 min (2,3-DCP) and 3.6 min (ECH).

Protocol2