Team:UESTC-China/notebook protocal

protocol

• bacteria
• Molecular Biotechnology
• Analysis
• Reagent

Bacteria

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 1. Competent E.coli Cells Preparation

1. Inoculate E.coli strains on culture plate without antibiotics and streak, place upside down and culture at constant temperature of 37°C overnight.
2. Select a well-grew colony using a sterile toothpick and inoculate it into 20ml LB medium with no antibiotics, culture at 37°C overnight while shaking (180rmp).
3. Pipet 1ml bacterium solution into 100ml LB liquid medium without antibiotics, incubate at 37°C for 1 hour and 40 minutes while shaking, at 180rmp, making OD600 is 0.4—0.6.
4. Transfer the culture solution into a 50ml BD tube, incubate on ice for 10 minutes.
5. Precool the centrifuge in advance and centrifugation at 4°C、4000rmp for 10 minutes.
6. Discard the supernatant, add 16ml pre-cooled 0.1M CaCl2 into a centrifuge tube, pellet cells and re-suspend, then incubate on ice for 10 minutes, and centrifugation at 4°C、4000rmp for 10 minutes.
7. Discard the supernatant, add 1.5ml pre-cooled 0.1M CaCl2 with 15% glycerol, re-suspend and incubate on ice for 10 minutes.
8. Add aforesaid suspensions into pre-cooled 1.5ml centrifuge tubes with 100ul per tube, freeze at -80°C.

 

 2.Overnight Cultures

1. Add 6mL autoclaved LB in a 50ml BD bottle.
2. Pipet 2μL of 1000X antibiotic into the LB.
3. Select a single colony using a sterile toothpick or pipette tip.
4. Place toothpick or pipette tip in the culture tube and stir.
5. Remove toothpick, or leave in the bottle.
6. Place culture tube in incubator at 37°C overnight shaking vigorously (180 RPM).

 

 3.Plates Screening

1. Pour 20mL autoclaved LB with appropriate antibiotic into each plate.
2. Take the appropriate amount of bacterium suspension on the plate and spread it homogeneous.
3. Seal the plate with parafilm, place upside down and culture at constant temperature of 37°C for 12 – 14 hours.

 

 4.Bacteria Preservation

1. Mix 900ul with 900ul 40% glycerol
2. store at -20℃

 

Molecular Biotechnology

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We used different molecular biological methods in our project. All used methods are listed below.

 1.PCR

In our project, in order to be able to finally amplify the target fragments. we try the following PCR systems and the procedure settings.

KOD amplification system

Component	50ul Reaction
10× KOD buffer	5ul
2mM dNTPs	5ul
25mM MgSO4	3ul
10uM forward primer	1ul
10uM Reverse primer	1ul
Temp	1ul(1ng/l)
KOD enzyme	1ul
ddH2O	33ul

KOD PCR program

Segment	Cycles	Temperature	Time
Initial denaturation	1	94°C	3min
Denaturation	38	94°C	30s
Renaturation	38	56°C	30s
Prolongation	38	68°C	30s/kb
Terminal prolongation	1	68°C	3min
heat preservation	1	12°C	10min

I-5 2X High-Fidelity Master Mix

Component	50ul Reaction
I-5 Mix	25ul
10uM forward primer	2ul
10uM Reverse primer	2ul
Temp	1ul(1ng/l)
ddH2O	25ul

I-5 PCR program

Segment	Cycles	Temperature	Time
Initial denaturation	1	98°C	2min
Denaturation	37	98°C	10s
Renaturation	37	65°C	10s
Prolongation	37	72°C	15s/kb
Terminal prolongation	1	72°C	5min
heat preservation	1	4°C	Hold

Q5 amplification system

5×Q5 buffer	10ul
10mM dNTPs	1ul
10uM forward primer	2.5ul
10uM Reverse primer	2.5ul
Temp	1ul(1ng/l)
Q5 enzyme	0.5ul
5×Q5 high GC Enhancer	10ul
ddH2O	22.5ul

Q5 PCR program

Segment	Cycles	Temperature
Initial denaturation	1	98°C
Denaturation	35	98°C
Renaturation	35	56°C
Prolongation	35	72°C
Terminal prolongation	1	72°C
heat preservation	1	12°C

Fusion PCR System

Component	50l Reaction
10× KOD buffer	5l
2mM dNTPs	5l
25mM MgSO4	3l
forward primer	1l
Reverse primer	1l
fragment 1	the moles rate of fragment1 to fragment2 is 1:1
fragment 2	the moles rate of fragment1 to fragment2 is 1:1
KOD enzyme	1l
ddH2O	to 50l

Fusion PCR Program

Segment	Cycles	Temperature
Initial denaturation	1	94°C
Denaturation	38	94°C
Renaturation	38	56°C
Prolongation	38	68°C
Terminal prolongation	1	68°C
heat preservation	1	12°C

Colony PCR MasterMix

Component	25ul Reaction
10×buffer	2.5ul
10mM dNTP	0.5ul
10uM Primer 1	0.5ul
10uM Primer 2	0.5ul
Temp	5ul
Taq enzyme	0.4ul
ddH2O	15.6ul

Colony PCR Program

Segment	Cycles	Temperature
Initial denaturation	1	95°C
Denaturation	38	94°C
Renaturation	38	56°C
Prolongation	38	72°C
Terminal prolongation	1	72°C
heat preservation	1	12°C

 2.Agarose Gel

Agarose Gel Preparation

1. Weight 0.3g agarose, mix with 30ml 1x TAE in a conical flask (0.5g, 50ml for long dies).
2. Heat the flask in the microwave oven until the solution becomes transparent, cool down to 40°C.
3. Add 0.1ul EB into solution and mix evenly by shaking slightly (2.5l EB for 50ml TAE)
4. Pour the solution into a die and wait for the gel solidifying.

 

Agarose Gel Electrophoresis

1. Add 5ul(10ul) loading buffer into a 25ul(50ul) PCR product.
2. Apply onto agarose gel together with makers.
3. Run at 130V for 25 minutes for a full gel.

 

Gel Extraction of DNA(Omega Gel Extraction Kit)

1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube.
2. Add a 3x sample volume of Buffer DE-A.
3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved. Intermittent vortexing to accelerate gel solubilization.
4. Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol.
5. Place a Miniprep column into a 2 ml microfuge tube. Transfer the solubilized agarosefrom Step 4 into the column. Centrifuge at 12,000xg for 1 minute.
6. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.
7. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds.
8. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute.
9. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute.
10. Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided). To elute the DNA, add 25 μl of deionized water（65℃） to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute.

 

 3.Ligation

T4 ligation system

Component	20ul Reaction
10× Ligation buffer	2ul
T4 Ligation enzyme	1ul
Fragment of digestion	the moles rate of insert DNA to vector DNA is 5:1 to 10:1
Vector of digestion	the moles rate of insert DNA to vector DNA is 5:1 to 10:1
ddH2O	to 20ul

Incubate at 22℃ for 2 hour

T4 ligation system

Component	20ul Reaction
10× Ligation buffer	2ul
T4 Ligation enzyme	1ul
Fragment of digestion	the moles rate of insert DNA to vector DNA is 5:1 to 10:1
Vector of digestion	the moles rate of insert DNA to vector DNA is 5:1 to 10:1
ddH2O	to 20ul

Incubate at 50℃ for 1 hour

 4.Transformation

1. Pipet each ligation product into a 1.5 mL tube with 100uL competent E.coli TOP10
2. Put on ice for 20 minutes
3. Incubate at 42°C for 90 seconds
4. Put it back on ice for 3 minutes
5. Add 500l LB media
6. Incubate for 1 hour at 37°C and 220 rpm
7. Centrifuge tubes for 2 minutes at 6500 rpm
8. Pipet 500uL supernatants out , pipet up and down the left liquid to suspend the cells
9. Spread plate with glass bead
10. Culture in an incubator overnight (18 hours) at 37°C

 

 5.Confirmation

Colony PCR

In order to detect if target fragments are successfully transformed into Escherichia coli, we choose colony PCR as the primary method to detect positive colonies, in order to improve the accuracy, we select primers in vectors as primers colony PCR. The detailed colony PCR system and PCR Program setting are in NO.2 PCR.

 

Plasmids extraction

1. Collect 5 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the bacteria.
2. Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing.
3. Add 250 μl of Buffer S2, and mix by gently inverting the tube for 7×.
4. Add 350 μl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000×g for 10 minutes to clarify the lysate.
5. Place a Miniprep column into an uncapped 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000×g for 1 minute.
6. Pipette 500 μl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
7. Pipette 700 μl of Buffer W2 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
8. Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column again and centrifuge at 12,000×g for 1 minute.
9. Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000×g for 1 minute.
10. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube. Add 50μl deionized water（65℃）to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.

 

Enzyme digestion

In order to further validate the accuracy of the plasmid obtained from the positive clones selected by colony PCR, we select the double enzyme digestion for second verification.

1.system

Component	50l Reaction
10× FD Green buffer	5ul
Forward enzymes	1ul
Reverse enzymes	1ul
Temp	1ng
ddH2O	to 50ul

2.Put it in 37℃ Constant temperature incubator for 1h

3.DNA Electrophoresis（No loading buffer）

 

Sequencing

Take plasmids 10ul that prove right with restriction enzyme and corresponding primers and send to sequencing company for finally confirming.

 

Analysis

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 1.SDS-PAGE

Make the separating gel

1. Set the casting frames on the casting stands.
2. Prepare the gel solution(as described above) in a separate small beaker.
3. Swirl the solution gentlybut thoroughly.Pipet appropriate amount of separatinggel solution (listed above) into the gap between the glass plates.
4. To make the top of the separating gel behorizontal, fill in isopropanol into the gap until overflowing.
5. Wait for 1h to let itgelate.

 

Make the stacking gel

1. Discard the isopropanol and you can see separating gel left.
2. Pipet in stacking gel untill a overflow.
3. Insert the well-forming comb without trapping air under the teeth. Wait for 40min to let it gelate.

 

Take the galss plates in the electrophoresis bath

1. Make sure a complete gelation of the stacking gel and take out the comb.
2. Take the glass plates out of the casting frame and set them in the cell buffer dam.
3. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring
4. after overflow untill the buffer surface reaches the required level in the outer chamber.

 

Prepare the samples

1. Mix your samples with 5X lodaing buffer.(160L sample + 40L loading buffer).
2. Heat them in boiling water for 5 min.

 

Sample loading

1. Load protein marker in to the first lane.
2. Load prepared samples into wells and make sure not to overflow.
3. Then cover the top and connect the anodes.

 

Run

1. Set 80V before the downmost sign of protein marker reach the separating gel, it takes about 0.5h.
2. Change the volt to 120V when the downmost sign of protein marker reach the separating gel, It takes about 0.5h.

 

Dying and bleaching

1. You can stop running when the downmost sign of the protein marker almost reaches the foot line of the glass plate.
2. Take out the separating gel carefully, and dye in the coomassie brilliant blue solution(as described above) for 6h.
3. Take out the separating gel and rinse it twice by UP water.
4. Bleach it by destaining solution for 1.5h.

2. Enzyme activity detection

Congo red assay

Luria agar plates with 0.2% CMC are inoculated with the strain and incubated at 37°C for 24h. Next day the agar is flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution is poured off into a toxic waste bottle and 1 M NaCl is added and left for another 1 h.Then pour off NaCl solution.

3,5-dinitrosalicylic acid (DNS) method

Reactions are carried out in 50 ml centrifuge tubes with substrate in the buffer, plus enzyme solution. Reaction mixtures are incubated at 40 °C for 3 h, enzyme action is interrupted by the addition of 3,5-dinitrosalisylic acid (DNS) reagent, which is used to quantify the total amount of reducing sugars. Reaction mixtures are then placed in a boiling water bath for 5 min, cooled to room temperature and diluted to 25 ml with water for the measurement of absorbance at 540 nm with a spectrophotometer.

 

Reagent

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1.Luria-Bertani Liquid Medium (1L)

Components	Contents
typtone	10g
yeast extract	5g
NaCl	10g

2.Luria-Bertani Solid Medium (1L)

Components	Contents
typtone	10g
yeast extract	5g
NaCl	10g
agar	12g

3.Antibiotics

1000× Kanamycin(50mg/mL)	1000× Kanamycin(50mg/mL)	1000× Ampicillin(100mg/mL)	1000× Ampicillin(100mg/mL)
Kanamycin	ddH2O	Ampicillin	ddH2O
1g	20ml	2g	20ml

4.40% glycerol

Components	Contents
glycerol	40ml
ddH2O	60ml

5.50×TAE

Components	Contents
Tris base	242g
Na2EDTA∙2H2O	37.2g
ice vinegar	57.1mL
ddH2O	800mL

6.SDS-PAGE

Gel

	Separating Gel(12%)	Stacking Gel(5%)
ddH2O	3.35mL	3.4mL
30% Acrylamid	4ml	830mL
1.5M Tris	2.5ml (PH=8.8)	430mL (PH=6.8)
10% lauryl sodium sulfate	100mL	50mL
10%ammonium	40mL	50mL
peroxydisulfate
tetramethylethylenediamine	10mL	5mL

Other

5X loading buffer	1mol/L Tris-HCl(pH6.8) 2.5mL Glycerol 5mL lauryl sodium sulfate 1 g Bromophenol blue 50mg β-2- mercaptoethanol 250uL ddH2O 2.5mL
5X SDS-PAGE running buffer	Tris 15g， Glycine 94g， lauryl sodium sulfate 5g， Add ddH2O to 1L， Diluted five times while using.
Commassie BlueStaining Solution	Commassie Blue R250 0.25g Carbinol 45mL Glacial acetic acid 10mL Add ddH2O to 100mL
destaining solution	Carbinol 45mL Glacial acetic acid 10mL Add ddH2O to 100mL

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