Team:WHU-China/Notebook/Protocols

Protocols

•           Dealing with biobricks

•           Preparation of competent cells (Using Calcium Chloride)

•           Plasmid Extraction（Using Plasmid Mini Kit）

•           Transformation (heat-shock method)

•           Cleavage with two restriction enzymes

•           Get Extraction

•           Identification of DNA by Electrophoresis

•           Ligation

•           PCR of the colony

•           Identification of DNA by Sequencing

•           Others

Ⅰ.Dealing with biobricks

1.1            Storage

1.        Dried DNA: room temperature

2.        Resuspended DNA: -20℃ freezer

3.        The linearized plasmid backbones (25ng/ul at 50ul) should be stored at 4C or lower

1.2            Usage

1.        With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.

2.       

3.        Add 10uL of diH2O (deionized water), the resuspension will become red due to the cresol red dye used during manufacturing.

4.        Pipette 1 or 2uL of the resuspended DNA transform into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.

5.        Pick a single bacterial colony and transfer it into LB medium. Incubate the culture for 18 hours with vigorous agitation.

6.        Store the single colony in physiological saline and on inclined plane, and make recognizable marks on the tube and on the notebook.

Ⅱ. Preparation of competent cells (Using Calcium Chloride)

2.1  Day 0:

    Pick a single bacterial colony from a plate that has been incubated for 16-20 hours at 37℃. Transfer the colony into LB medium. Incubate the culture overnight (about 16 hours) at 37℃ with vigorous shaking.

2.2  Day 1:

1.        Transfer 1 ml of the culture into 100 ml of LB medium. Incubate the culture for 2.5-3 hours at 37℃ with vigorous agitation (250-300rpm).

2.        Transfer 1.5 ml of the culture into sterile ice-cold 1.5ml polypropylene tubes. Cool the cultures by storing the tubes on ice for 10 minutes.

3.        Recover the cells by centrifugation at 2500 rpm for 5minutes at 4℃.

4.        Decant the medium from the cell pellets. Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M CaCl2 solution. Store the tubes on ice for 20minutes.

5.        Recover the cells by centrifugation at 2500 rpm for 5 minutes at 4℃.

6.        Decant the medium from the cell pellets. Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M CaCl2 solution.

7.        The suspension of competent cells should be immediately used in transformation.

 

Attention:

[1]     Mix gently

[2]     The newly prepared competent cells should be transformed immediately. They cannot be stored for long (except in -80℃)

[3]     Keep sterile environment during operation (sterilize the clean bench by ultra-violet, sterilize hands and implements by alcohol).

[4]     Use heat-shock method to transform.

 

Ⅲ. Plasmid Extraction（Using Plasmid Mini Kit）

3.1  Things to do before starting:

•         Preheat Elution Buffer to 70°C if Plasmid DNA is >10kb

•         Dilute DNA Wash Buffer with absolute ethanol and Add vial of RNase A provided to Solution I.

3.2  Details:

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).

2.        Decant or Pellet bacterial cells by centrifugation at 10,000 x g for 1 min at room temperature.

3.        Resuspend the bacterial pellet by adding 250 μl of Solution I/RNase A solution, and vortexing (or pipetting up and down). Complete re-suspension (no visible cell clumps) of cell pellet is vital for obtaining good yields. Transfer suspension into a new 1.5 ml microcentrifuge tube.

4.        Add 250 μl of Solution II and gently mix by inverting and rotating tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity.

Note: Do not allow the lysis reaction to proceed more than 5 min.

5.        Add 350 μl of Solution III and mix immediately by inverting 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.

6.        Centrifuge at 13,000 x g for 10 min at room temperature. A compact white pellet will form. Promptly proceed to the next step.

7.        Prepare a HiBind DNA Mini Column by placing into a 2 mL collection tube.

8.        Add 100 μl of Equilibration Buffer. Centrifuge at 13,000 x g for 30-60 seconds.

9.        Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.

1)        Add the cleared supernatant from step 6 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Ensure that the pellet is not disturbed and that no cellular debris has carried over into the HiBind DNA Mini Column. Centrifuge at 13,000 x g for 1 min at room temperature to completely pass lysate through the HiBind DNA Miniprep Column. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.

2)        Add 500 μl of HB Buffer and centrifuge at 13,000 x g for 30 to 60 seconds at room temperature to wash the HiBind DNA Mini Column. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube. This step ensures that residual protein contaminations are removed, thus ensuring high quality DNA that will be suitable for downstream applications.

3)        Add 700 μl of DNA Wash Buffer (diluted with absolute ethanol) and centrifuge at 13,000 x g for 30 to 60 seconds at room temperature to wash the HiBind DNA Column. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.

NOTE: DNA Wash Buffer Concentrate must be diluted with absolute ethanol before use.

10.    OPTIONAL: Repeat wash step 10 with another 700 μl of DNA Wash Buffer (diluted with absolute ethanol).

11.    Centrifuge the empty HiBind Mini Column at 13,000 x g for 2 min to dry the column

IMPORTANT: Do not skip this step - it is critical for good yields

12.    Place the HiBind DNA Mini Column into a new/clean 1.5 ml microcentrifuge tube (not supplied). Depending on desire concentration of final product, add 30-100 μl of Elution Buffer (10 mM Tris-HCl, pH 8.5) or sterile deionized water directly onto the center of the column matrix. Incubate at room temperature for 1 minute. Centrifuge for at 13,000 x g for 1 min to elute DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

13.    Yield and quality of DNA: Determine the absorbance of an appropriate dilution of the sample at 260 nm and then at 280nm.

Ⅳ. Transformation (heat-shock method)

1.        Add DNA (Plasmid: 1ul; Ligation: all) to each tube in which 100ul newly prepared competent cells are stored. Mix the contents of the tubes by swirling gently. Ensure that the competent cells are stored on the ice all the time.

2.        (Negative control: 50uL DH5αcompetent cells+1uL sterile H2O)

3.        Store the tubs on ice for 30 minutes.

4.        Heat shock：Transfer the tubes to a rack placed in a preheated 42℃