Preparation of LB medium

LB (Lysogeny Broth) Culture	1L
Tryptone	10g
Yeast Extract	5g
NaCl	10g
Agar	15g (1.5%)

Pour 10 plates, need to add Kana before pouring, 100ml one 100ul Kana

250ml for solid, 250ml for liquid

Simultaneous sterilization: water, two boxes of small, medium and large gun tips, 1.5 ml tubes

Diamond Plasmid Mini Prep Kit

1.  Preparation

•  Add the RNase A to Buffer SP1.

•  Add the volume of 100% ethanol indicated on the bottle label to the Wash Solution.

•  Check Buffer SP2 and SP3 for precipitation.

1.  Equilibrate column: Place a Miniprep column into an collection tube. Add 500 ul Buffer S to column, centrifuge at 12,000 X g for 1 min. Discard the filtrate in collection tube. Return the column to the collection tube.

2.  Collect 1.5-5ml of overnight LB bacteria culture. Centrifuge at 8,000 X g for 2 min to pellet the bacteria. Discard as much of the supernatant.

3.  Resuspend the bacterial pellet in 250plof Buffer SP1 by vortexing. Make the pellet completely resuspended.

4.  Add 250 pl of Buffer SP2, and mix by gently inverting the tube for 5-10 times. And incubate at room temperature (15-25 C) for 2-4 min.

5.  Add 350pl of Buffer SP3, and mix immediately by gently inverting the tube for 5-10 times.

6.  Centrifuge at 12,000 X g for 5-10 min. Transfer the supernatant to column, centrifuge at 8,000 X g for 30 sec, discard the filtrate in the collection tube.

7.  (Optional) Add 500pl Buffer DW1, Centrifuge at 9,000 X g for 30 sec, discard the filtrate in the collection tube. Return the column to the collection tube.

8.  Add 500pl Wash Solution, centrifuge at 9,000 X g for 30 sec, discard the filtrate in the collection tube. Ret turn the column to the collection tube.

9.  Repeat step 9.

10.  Place the column back into the collection tube. Centrifuge at 9,000 X g for 1 min.

11.  Transfer the column into a clean 1.5 ml Eppendorf tube. To elute plasmid, add 50~100pl of Eluent buffer to the centre of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 X g for 1 min. Keep the DNA solution.

Enzymatic digestion

Enzymatic cleavage of target fragments and vector:

1.  Ndel/Xhol double digested target fragments: ①Asp, ②Sga, ③Sva, 900~1000bp

2.  Ndel/Xhol double digestion pET-28a(+) plasmid, about 6000bp.

Purpose fragment 3 samples, make two copies of each, total 6 samples. Plasmid 3 samples. Total 9 samples.

Reaction conditions: 37℃/30min

Gel Electrophoresis

Reagents	Volume
Agarose	1.5% - 1.5g
TBE	100ml
Loading Buffer per sample	2µl
DNA Marker	2ul

Gel Extraction - Diamond DNA Gel Extraction Kit

1.  Preparation: before use the kit:

–  Check Wash Solution for addition of 100% ethanol.

–  Check Buffer B2 for precipitation.

–  Adjust water bath to 50'C.

2.  Excise the agarose gel slice containing the DNA fragment of interest and weight.

3.  Add a 3-6 times of sample volume of Buffer B2, heat at 50'C in water both for 5-10 min until the gel is completely dissolved.

4.  (Optional) If the DNA fragments is less than 500 bp, add further with 113 volume of isopropanol.

5.  Transfer the binding mix to the column and centrifuge at 8000 X g for 30 sec. Discard the filtrate in the collection tube. Return the column to the collection tube.

6.  Add 500 pl Wash Solution to column and centrifuge at at 9,000 X g for 30 sec, Discard the filtrate in the collection tube. Return the column to the collection tube be.

7.  Repeat step 6.

8.  Place the column back in the collecting tube and centrifuge at 9,000X g for 1 min.

9.  Place the column back in a new 1.5 ml Eppendorf tube, add 15-40 pl Elution Buffer to the center of the column membrane, after sitting at room temperature for 1 min, centrifuge for 1 min. Keep the DNA elution.

Ligation Protocol with T4 DNA Ligase (M0202) NEB

1.  Setup the following reaction in a microcentrifuge tube on ice

  (T4DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.

COMPONENT	20ul REACTION
T4 DNA Ligase Buffer (10x)*	2 ul
Vector DNA (4 kb)	50 ng (0.020 pmol)
Insert DNA (1 kb)	37.5 ng (0.060 pmol)
Nuclease-free Water	Up to 20 ul
T4 DNA Ligase	1 ul

*The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

1.  Gently mix the reaction by pipetting up and down and microfuge briefly.

2.  For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

3.  For blunt ends or single base overhangs, incubate at 16 overnight or room temperature for 2hours (alternatively, high concentration T4 DNALigase can be used in a 10minute ligation).

4.  Heat inactivate at 65C for 10 minutes.

5.  Chill on ice and transfom 1-5μl of the reaction into 50μl competent cells.

Culturing Bacteria

Total 3 samples ①Asp-pET28a, ②Sga-pET28a, ③Sva-pET28a

Reaction conditions: 16°C/2h (overnight is best), 37°C/30min

Inactivation by heating at 65°C for 10 min

Conversion of DH5α by enzyme-linked products

Experimental design

1.  3 samples were transformed: ①Asp-pET28a, ②Sga-pET28a, ③Sva-pET28a.

2.  Coat on 3 Kana/LB plates (3 plates are required).

3.  37℃ overnight incubation culture

Transformation steps:

1.  Remove 100ul load of E. coli receptor cells from -80℃ refrigerator and thaw on ice:

2.  Add 5ul of recombinant product, shake gently and leave on ice for 10min;

3.  42℃ water bath for 90s, rapidly transfer to ice for 2min;

4.  In the ultra-clean bench: add 700ul LB liquid medium to the EP tube, mix well with the bacterial solution, and resuscitate at 37℃ for 1h;

5.  the bacterial solution coated with antibiotic-containing LB plate (prepared in advance or on-site inverted plate), until the bacterial solution air-dried inverted culture, 37 ℃ overnight (about 16h)

Preparation of IPTG

1.  IPTG: isopropyl-β-D-thiogalactopyranoside, molecular weight 238.3, a kind of strong inducing agent, not metabolized by bacteria and very stable.

  Preparation of storage solution: Dissolve 1g of IPTG in 4ml of distilled water, then dilute to 5ml with distilled water, sterilize with a disposable needle filter, and then divide into 1ml small portions for storage at -20℃. (Concentration is 0.8mol/l)

Pick BL21 plate monoclonal shaking bacteria

I. Pick DH5α plate monoclonal shaking bacteria

Induction of protein expression

1.  Transfer the bacterial solution to LB medium at 1% and incubate at 37℃ for 4h. 2.

2.  When the OD600 of the bacterial solution reaches 0.6, remove and cool the shake flask, then add 0.8MIPTG in the proportion of 0.2% to induce protein expression, and then incubate at 16℃ for 20 hours.

Purification reaction application proteins:

1.  ultrasonic breaking of bacteria

1.  Sterilization: pre-cooled to 4 degrees using a cell breaker

2.  lubricate two times, the first time with water and the second time with Buffer before feeding.

3.  Inject the sample, circulate for a while and then pressurize it to about 700-800 bar, and calculate the running time.

4.  After the end of the run, rinse five times, except for the third time with 20% anhydrous ethanol, the rest of the time with water.

2.  Centrifugation

1.  pre-cool to 4°C

2.  centrifuge leveling +/- 0.06g

3.  run at 9500 rpm for one hour

3.  Nickel column purification

1.  Equilibrate the column: add 20 column volumes or more of Buffer A to the column.

2.  Ultrasonically crush the supernatant for two minutes to break up the residual DNA.

3.  Take 0.45 µL of sample, syringe through the membrane and onto the nickel column.

4.  Collect the filtrate and repeat step 3.

5.  Buffer A washes out the non-specific binding proteins (about 20 column volumes).

6.  Buffer B elutes specifically bound proteins (use gradient elution when doing this for the first time)

7.  Post-treatment

4.  Anion-exchange column chromatography to elute DNA-binding proteins.

1.  Decontamination: wash the adsorbent with 1M NaCl (column volume 5ml). 2.

2.  Wash the adsorbent with three times the column volume of water. 3.

3.  Equilibration: Wash the adsorbent with three times the column volume of Buffer.

4.  Sampling: take a sample before and after the sample passes through the column (if there is only DNA on the adsorbent, leave out the liquid as the sample; if there is still protein on the adsorbent, use FPLC to elute the protein).

5.  Post-treatment: Wash the column with 1M NaCl solution at 2-3 times the volume of the column and water at 4 times the volume of the column.

5.  Heparin adsorption (DNA removal)

1.  Equilibrate the column with four times the column volume of Buffer. 2.

2.  Dilute the sample using salt-free buffer

3.  Sample passes through heparin adsorption column (test for hang-ups, if no hang-ups then need to continue diluting sample)

4.  FPLC

Nickel column chromatography

Buffer A

COMPONENT	PARAMETER
MES - pH6.7（Replaceable with Tris-Cl - pH 8.0）	20 mM
NaCl	300 mM
Glycerol	5%
Imidazole	50mM

Buffer B

COMPONENT	PARAMETER
MES - pH6.7（Replaceable with Tris-Cl - pH 8.0）	20 mM
NaCl	300 mM
Glycerol	5%
Imidazole	500mM

Heparin adsorption column ( clear DNA )

Buffer A

COMPONENT	PARAMETER
Tris-Cl - pH 8.0	20 mM
NaCl	50 mM
Glycerol	5%

Buffer B

COMPONENT	PARAMETER
Tris-Cl - pH 8.0	20 mM
NaCl	1 M
Glycerol	5%

Diluted Buffer

COMPONENT	PARAMETER
Tris-Cl - pH 8.0	20 mM
Glycerol	5%

Desalting (similar to molecular sieve)

1.  Equilibrate the column: Wash the column with four times the volume of Buffer.

2.  Add the remaining liquid from the upper portion of the ultrafiltration tube to the adsorption column.

1.  if the volume of liquid is greater than 2.5 ml, aspirate 2.5 ml

2.  if the volume of liquid is less than 2.5 ml, aspirate liquid from the lower part and replenish to 2.5 ml

3.  3.5 ml of buffer is added to the column and the liquid is collected in a buffer-wetted ultrafiltration tube.

4.  Post-treatment: the column is washed 4 times with water and stored.

Concentration (ultrafiltration, about 1 ml)

1.  Ultrafiltration tubes are rinsed with buffer.

2.  centrifuge at 4500 rpm

3.  after concentration, transfer the liquid from the upper tube to a small blotting tube, freeze and centrifuge at 12,000 rpm for 5-20 minutes, and transfer the sample to a new blotting tube.

4.  Post-processing: Use 0.1M NaOH for 15-30 minutes and soak in water and store in 20% ethanol.

Protein Preservation

•  Add 50% glycerol to half of the proteins and store at -40 degrees Celsius in 50 ul tubes.

•  Add 5% glycerol to half of the proteins and store 50ul per tube at -80 degrees Celsius.

Protein Quantification and Concentration Detection

Plotting the standard curve

1.  For 300 microliters of the system, 285 microliters of Bradford's solution is required, and for the other fifteen microliters, 0-6 microliters of 1mg/ml BSA is added sequentially to the system and the volume is replenished to 15 microliters by adding buffer.

2.  Mix for 5 to 10 minutes at room temperature.

3.  Absorbance at 595 nm was measured spectrophotometrically.

Protein samples

1.  Take three dilutions, as dilute as possible, and measure more than one value.

2.  Place the plate in the enzyme counter.

Protein Function - Binding and Cleavage Test

Detection of DNA composition using agarose gel electrophoresis

1.  use DNA extracted from BL 21 and b7a strains, where b7a, the bacterium, carries natural sulfur-modified DNA.

2.  Set the voltage to 120 volts and electrophoresis the gel for 40 minutes.

Configure 10 microliters of system sample

5x Buffer	DNA (5pµ/µl)	Protein (15pµ/µl)	ddH2O
2	1	0	7
2	1	0.3	6.7
2	1	0.6	6.4
2	1	1.2	5.8
2	1	2.4

5x Buffer: 1ml Tris-Cl and 0.5ml NaCl

1.  Add 0.5µl marker

2.  Add 5µl sample

3.  Use 15µA current and carry out electrophoresis for 40 minutes

Perform a gel blocking assay (mini-gel electrophoresis) to check if the protein is bound to the DNA.

Reagent	Volume/µl
Tris (pH 6, 6.5, 7)	20
NaCl	2
DTT	2
MnCl2	2
ddH2O	74

If the protein binds to the DNA, the total volume becomes larger and moves closer in the gel

Configuration of cutting experiment buffer

Configure Cutting Experiment Sample (10µl)

Reagent	Volume/µl
2x Buffer	5
DNA (150ng/µl)	2
Protein	1
ddH2O	2

1.  set the metal bath at 37 degrees Celsius and wait for one hour for cutting.

2.  2 (control) x 3 (pH=6, 6.5, 7) x 6 (protein concentration) = 36 samples were produced.

3.  1µl of proteinase k was added to degrade the protein so that the gel electrophoresis results were not affected by the protein.

4.  set the metal bath at 50 degrees Celsius and wait for 30 minutes to degrade the proteins.

5.  0.5µl was added and gel electrophoresis was carried out at 120V for 40 minutes.

Vazyme Biotech Co.,Ltd. Gram-Negative Bacteria DNA Extraction

Experimental Procedure

Sample Processing for E.coli - Gram-negative bacteria

1.  Take 1-5 ml of bacterial culture (less than 1.0 x 10% bacteria), centrifuge at 10,000 rpm (11,500 x g) for 1 min, and pour off the culture.

–  The number of bacteria can be measured by spectrophotometer, 10D60 is about 1.5×10° bacteria.

2.  Add 230 μl BufferGA and shake until the bacteria are thoroughly suspended.

3.  Add 20μulProteinaseK and shake to mix.

4.  Add 250ulBufferGB, shake and mix well, 70℃C water bath for 10min.

–  The addition of BufferGB may produce white precipitate, which will disappear at 70C and will not affect the subsequent experiments. If the solution does not become clear, it means that the cell lysis is not complete, which may result in a small amount of extracted DNA and impurity.

5.  (Optional) If the RNA residue has a big influence on the subsequent experiments, add 4μl RNaseA to the digestion solution, shake for 15sec, and leave it at room temperature for 5-15min.

6.  Enter 08-2 for column purification.

Vazyme Biotech Co.,Ltd. DNA Purification

1.  Add 180μl of anhydrous ethanol, shake and mix well, flocculent precipitate may appear, centrifuge briefly to collect the liquid on the inner wall of the cap.

2.  Transfer the above mixture to a FastPuregDNAMini Columns Ill adsorbent column (adsorbent column has been placed in the collection tube) and centrifuge at 12,000 rpm (13,400*g) for 1 min. run the filtrate.

3.  Add 500ul of BufferPB (please check that anhydrous ethanol has been added before use) to the adsorbent column and centrifuge at 12,000 rpm (13,400*g) for 1min, discard the filtrate.

4.  Add 600μl BufferPW (please check whether anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12,000rpm (13.400×g) for 1min, discard the filtrate.

5.  Repeat step 4.

6.  Put the adsorption column back into the collection tube and centrifuge the empty tube at 12,000 rpm (13,400 × g) for 2 min.

–  After centrifugation of the empty column, it can be left uncapped for 2-5min to allow the residual ethanol to evaporate completely.

7.  Transfer the adsorbent column to a new 1.5 ml centrifuge tube (supplied), add 50-100 u of Elution Buffer to the center of the adsorbent column membrane, leave it at room temperature for 2-5 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min.

–  Note: The following steps can all help to increase DNA yield.

–  Preheat the ElutionBuffer to 55 before eluting;

–  Repeat the elution step with new Elution Buffer (this procedure increases the yield but decreases the concentration);

–  To increase the concentration of DNA, reintroduce the solution from the first elution to the adsorption column for elution.

8.  Discard the column and store the DNA product at -30 to -15°C to prevent degradation.