1.Preparation of liquid LB solution for competent cell culture and plasmid extraction
Ingredients
100ml
250ml
NaCl
1g
2.5g
Tryptone
1g
2.5g
Yeast
1g
2.5g
2. Preparation of solid LB solution for E. Coli competent cell culture petri dishes
Ingredients
100ml
250ml
NaCl
1g
2.5g
Tryptone
1g
2.5g
Yeast
0.5g
1.25g
Agarose
1.5g
3.75g
Kana Antibiotic
100μl
250μl
3. Preparation of Agarose Gel
1.Fold a weighing paper
2.Place the folded weighing paper on an electronic balance, press ‘peeling’ to zero the balance
3.Weigh the corresponded mass of agarose into the conical flask
4.Pour respective volume of TAE into the conical flask
5.Heat the solution with microwave for 3 minutes
6.Remove the conical flask from the microwave for inspection. If the agarose is completely dissolved, proceed to step 7. If the agarose is not completely dissolved, repeat step 5.
7.After cooling, add SybrSafe®(1/10000 v/v solution of the mixture) to the solution
8.Set up the gel mould
9.Gently pour the gel inside the mould, making sure the liquid is evenly spread
4. Polymerase Chain Reaction
1.Set up a PCR system (Always add the Polymerase last in case of mistake)
2.Place in PCR machine and set the program as needed
3.30min before PCR ends, make agarose gel for DNA recycling
4.Add 6x Loading Buffer into each system
5.The first lane of the left-hand side should always be the Marker (Trans2K DNA Marker)
PCR System (50μl)
Ingredients
Volume
5*Prime Star Buffer
10 μl
dNTP
4μl
Forward Primer
1.5μl
Reverse Primer
1.5μl
DNA template
1μl
Prime Star Polymerase
1μl
ddH2O
31μl
PCR Program
Pre-Denaturing
98°C, 2 min
1 cycle
Denaturing
98°C, 15s
35 cycles
Annealing
55°C, 15s
35 cycles
Extending
68°C, 1kb/min
35 cycles
Further Extending
68°C, 5 min
1 cycle
5. Bacterial Colony Polymerase Chain Reaction
1.30min before PCR ends, make agarose gel for DNA recycle (see step 6 for further details)
2.Pick a single colony with gun tip and emerge it underneath the surface of the system. Blend multiple times
3.Pipette 1μL of PCR system on the blocks already drown on the replicate plate
4.Pipette 1μL of PCR system on the blocks already drown on the replicate plate
5.Add corresponded primers (Primer F & Primer R), 1μL each to the tubes
6.Centrifuge shortly with the microcentrifuge
7.Vortex blending
8.Place in the PCR machine and set the program as needed (See charts below for further details
6. Gel Electrophoresis
1.Emerge the gel in the TAE buffer, making sure the liquid covers all the wells
2.Make sure the wells are placed toward the cathode
3.Pipette 20% volume of 6x Loading buffer into the samples, blend with vortex
4.Always put a marker in the leftmost lane
5.Add sample that is well mixed with loading buffer into the gel, record the order on the lab note
6.Electrophoresis should last for 30min, 150v.
7.If Marker appears too short, run the gel for an additional 30 minutes with close monitoring
8.After electrophoresis, obtain an image with the gel inspection machine and smart phone
7. Recycling of PCR Products
1.Prepare a 1.5ml tube (Marked with the amplified sequence name)
2.Placed the excised gel fragment in a 1.5ml Eppendorf tube
3.Compress the gel on the dip of the tube using a microcentrifuge. Estimate gel volume
4.Pipette equal volume of Buffer PN into the tube, mix with vortex, and place the tube inside a 50 ̊C metal bath until the gel fragment is fully dissolved
5.Balance a CA2 spin column with 500μL Buffer BL during the time of gel
dissolving 8. Centrifuge the spin column for 1min at 12000rpm, discard the flow-
through
6.Apply the dissolved gel solution into the CA2 spin column, letting stand for 2min and centrifuging at 12000rpm for 1min
7.Collect all the flow-through back to the CA2 spin column, centrifuge at 12000rpm for 1min
8.Discard the flow-through and pipette 600μL of Buffer PW to wash the CA2 column. Let stand for 2min then centrifuge at 12000rpm for 1min, discard the flowthrough
9.Pipette again 600μL Buffer PW, let stand for 2min, 12000rpm for 1min, and discard the flow-through
10.Put back the CA2 spin column, centrifuge 12000rpm for 3min without adding anything to get rid of the buffer.
11.Apply the CA2 spin column to a new 1.5ml tube (already labeled with code on the lid, plasmid name on the body) and put inside a metal bath with lid open at 50 ̊C for 5min
12.Pipette 30μL of buffer EB to the silicon film of the CA2 spin column, let stand in a 50 ̊C metal bath with lid closed
13.Obtain DNA concentration with a microplate reader, note it on the body of the tube(under the name of plasmid) and date of extraction underneath that. Update into the strain list
8. Restriction Enzyme Reaction
1.Use Nanodrop to test for the concentration of the recycled PCR products
2.Obtain 1-2 μl of target fragment
3.Prepare the reaction system according to the table below
4.Incubate the system at 37°C for three hours
5.Gel Electrophoresis and recycling of the target fragment (See steps 6 and 7 for details)
Ingredients
Volume
NED Enzyme I
1μl
NEB Engyme II
1μl
Target Fragment
1-2μl
10*Cutsmart Buffer
5μl
ddH2O
41-42μl
9. Gibson Assembly
1.Using 5μL smart mix to construct a Gibson assembly system
2.Set PCR program as: 50 ̊C 20min 12 ̊C forever
3.Blend Gibson system with 100μL of competent cells, ice bath over 30 min (Allow thorough contact of DNA and cells, enabling DNA to enter the cell wall)
4.Heat shock at 42 ̊C for 90s (Cells swell at high temperature, allow thorough contact of the cell membrane and cell wall, enabling DNA to pass through the cell membrane into the cytoplasm)
5.Ice bath for 5min(Cell shrinks at low temperature, dragging DNA on the wall into the cell)
6.Pipette 200μL of all nutrient broth into the tube(operate inside the bench), shake the bacteria in a shaker at 37 ̊C for 1h30min
7.Spread the bacteria on plates. Observe after 16h~20h
10. Preparation of Petri Dishes for Competent Cell Cultur
1.Solid LB solutiono prepared
2.Carefully pour the solution into clean petri dishes in ventilated Cell Culture Hood
3.Allow the solution to dry over 30 minutes
4.Seal the petri dishes with tape
5.Store in 4 ̊C fridge, upside down to prevent accumulation of moisture
11. E.Coli Competent Cell Culture Transfection
1.Place 50μl of Trans5α competent cells culture onto ice
2.Add the reacted fragment-backbone mixture solution into the competent cell culture
3.Place the mixed solution onto ice for 30 minutes
4.Stimulate the solution in 42 ̊C water bath for 60 seconds
5.Place the solution onto ice for two minutes
6.Add 1ml of liquid LB without antibiotics in ventilated Cell-Culture Hood
7.Proceed with step 12
12. Incubation
1.Prepare and label an empty 15ml test tube
2.Pipette 400μl of liquid LB solution into the test tube
3.Pipette the bacteria solution into the test tube with liquid LB
4.Place the test tube into 37 ̊C incubation for 45 minutes
13. Plasmid Extraction
1.Isolate a single colony from a freshly streaked selective plate, and inoculate a culture
of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hr at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a® and JM109®.
2.Centrifuge at 10,000 x g for 1 minute at room temperature
3.Decant or aspirate and discard the culture media
4.Add 250 μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.
Complete resuspension of cell pellet is vital for obtaining good yields Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents section on Page 6.
5.Transfer suspension into a new 1.5 mL microcentrifuge tube
6.Add 250 μL Solution II. Invert and gently rotate the tube several times to obtain a
clear lysate. A 2-3 minute incubation may be necessary. Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.
7.Add 350 μL Solution III. Immediately invert several times until a flocculent white
precipitate forms. Note: It is vital that the solution is mixed thoroughly and
immediately after the addition of Solution III to avoid localized precipitation
8.Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet
will form. Promptly proceed to the next step
9.Prepare the vacuum manifold according to manufacturer’s instructions
10.Connect the HiBind® DNA Mini Column to the vacuum manifold
11.Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the
HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular
debris is transferred to the HiBind® DNA Mini Column
12.Turn on the vacuum source to draw the sample through the column
13.Turn off the vacuum
14.Add 500 μL HBC Buffer. Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 6 for instructions
15.Turn on the vacuum source to draw the buffer through the column
16.Turn off the vacuum
17.Add 700 μL DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 6
for instructions
18.Turn on the vacuum source to draw the buffer through the column
19.Turn off the vacuum
20.Repeat Steps 17-19 for a second DNA Wash Buffer wash step
21.Transfer the HiBind® DNA Mini Column to a 2 mL Collection Tube
22.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Note: It is important to dry the HiBind® DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications
23.Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube
24.Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane. Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent on
pH. If using sterile deionized water, make sure that the pH is around 8.5
25.Let sit at room temperature for 1 minute.
26.Centrifuge at maximum speed for 1 minute. Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
27.Store DNA at -20°C
13. SDS-PAGE.
1.Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis.
2.Add marker in the first lane on the left at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.
