Team:XMU-China/protocols

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protocols

Gel Extraction

1. Weigh a 1.5mL centrifuge tube for each DNA fragment to be isolated and record the weight.
2. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 Ml tube and weigh. Record the weight of the gel slice.
3. Add Bing Buffer BD at a ratio of 100μL of solution per 100mg of agarose gel slices.
4. Incubate the gel mixture at 55-65℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
5. After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 minute at room temperature.
6 Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the flow-through.
7. Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 minute at 12,000 rpm , then discard the flow-through.
8. Repeat step 7 again.
9. Centrifuge the Columns for an additional 3 min to completely remove residual wash buffer.
10. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
11. Place the spin column in a clean 1.5 mL microcentrifuge tube, and pipet 20 μL deionized water (pH is 8.0-8.5 and prewarm to 60℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.
12. Centrifuge for 1 minute at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at –20℃.

Reaction system of restriction endonuclease
                    Table 1  The digestion system

System1、2、3 and 4 are used for Standard BioBrick Assembly .System 5 and 6 are used for Restriction analysis. Digestion of sample: at least 500 ng DNA / 10 µL volume. Digest for 4 h at 37 °C, afterwards inactivated by adding 10x loading buffer and standing for 10 min at room temperature.

Standard BioBrick Assembly
•Digestion of insert: 2 μg~5 μg DNA / 100 µL volume, 10x H buffer, EcoRI, SpeI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.
•Digestion of vector: 2 μg~5 μg DNA / 100 µL volume, 10 x M buffer, EcoRI, XbaI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

Suffix Insertion
•Digestion of insert: 2 μg~5 μg DNA / 100 µL volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.
•Digestion of vector : 2 μg~5 μg DNA / 100 µL volume, 10x H buffer, SpeI, PstI. Digestion and inactivation. Clean up the vector.

Ligation
•After digestion and clean-up, the next step is ligation. ligation at 16℃ for 4h or at 4℃ for 16h. Table 2 is the system of ligation.
Table 2  Ligation system

Restriction analysis
•Pick one colony with a sterile tip and cultivation in 20mL LB for overnight at 37 ℃
•Isolation of Plasmid
•Digest BioBrick，the system of Restriction analysis refer to table1
•Gel electrophoresis：add 2.2 µL loading buffer to digestion mixture. An agarose concentration is 1 %.

50mg/mL Kanamycin
- 0.5g Kan, 10mL water, filter sterilize with millipore express membrane, freeze in aliquots

100mg/mL Amp
-1g Amp, 10mL water, filter sterilize with millipore express membrane, freeze in aliquots.

50mmol/L Arabinose
-0.1876g Arabinose, 25mL water, filter sterilize with millipore express membrane.

Purity Plasmid Miniprep

1. Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution
2. Resuspened the pelleted cells in 250 μL of the Resuspension Solution (Mixture with Solution I and RNasa A). Transfer the cell suspension to a centrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
3. Add 250 μL of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times ,rest for 1-2minutes at room temperature until the solution becomes viscous and slightly clear.
4. Add 350 μL of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.
5. Centrifuge for 10 min at 12,000rpm to pellet cell debris.
6. Transfer the supernatant to the supplied spin column by decanting. Avoid disturbing or transferring the white precipitate.
7. Centrifuge for 1 min at 12,000rpm. Discard the flow-through and place the column back into the same collection tube.
8. Add 500 μL of the Wash Buffer PB to the spin column. Centrifuge for 1minute at 12,000rpm and discard the flow-through. Place the column back into the same collection tube.
9. Add 500 μL of the Wash Buffer W to the spin column. Centrifuge for 1minute at 12,000rpm and discard the flow-through. Place the column back into the same collection tube.
10. Repeat the step 9 again.
11. Discard the flow-through and centrifuge for an additional 3 min to remove residual Wash Solution.
12. Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 μL Elution Buffer TE (prewarm to 60℃) directly to the center of the column without touching the membrane. Rest for 2 min at room temperature and centrifuge for 1 min at 12,000rpm.
13. Discard the column and store the purified plasmid DNA at -20°C.

Transformation

1 Preparation of Competent BL21
- Thaw an aliquot of cells (without any plasmid in them) on ice
- To 50 mL of sterile LB, add 100μL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as sterile as possible (i.e. autoclave all solutions and use sterile and brands)
- Grow cells in the shaker at 37℃ until they reach an OD = 0.3-0.4 at 600nm (1cm pathlength cell). This usually takes 1.5-2 hours.
- Ice down the LB with growing cells for 10 min.
- Aliquot into sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and let drain upside down on a paper towel for ~1 minute.
- Ice down sterile 100mM CaCl2 and 100mM MgCl2 solutions during centrifugation.
- Gently resuspend each pellet in 8mL 0.1M MgCl2 and 2mL 0.1 M CaCl2 and combine into 2 tubes (this should take 1-2 minutes per tube)
- Centrifuge 6000rpm for 10 minutes and discard supernatant.
- Resuspend each pellet on ice in 100μL 0.1M of ice cold CaCl2 and combine into one tube

2 Transformation
- Add 10μL of DNA. Swirl gently with pipette.
- Incubate tubes on ice for 20 minutes
- Heat pulse tubes in 42℃ water bath for 30 seconds.
- Incubate on ice for 2 minutes
- Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking.
- Spread 100uL and 50uL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37℃ (spread using beads).