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Molecular Cloning Protocols

Transforming Competent E.coli with the Plasmid:

We chose the biobrick K137076 to ligate to the pvdQ gene for the following reasons:

• Take out the competent E-coli cells from the -80 freezer. (Keep all tubes on ice).  

• Add 1uL of the plasmid DNA in 100uL competent cells (if from Kit). For transformation of ligated product, add 10uL of the ligated plasmid DNA in 100uL competent

  cells.

• Incubate on ice for 10 min.

• Place in water bath at 42°C for 90s.

• Place immediately back on ice for at least 2 min.

• Add 800uL LB broth. Incubate for 1hr at 37°C shaker.

• Centrifuge at 130rpm for 5min to remove supernatant. Re-suspend the pellet in about 10uL of supernatant.

• Spread the entire re-suspended pellet on ampicillin agar dishes.

• Incubate for 12-16 hours at 37 °C.

 

Notes:

- Colonies grown on plate were selected for colony PCR screening.

- Correct colonies were then cultured in 5 mL LB Broth with 0.5uL ampicillin for subsequent screening or mass culturing.

- Mass culturing involved adding 200uL of the culture into 35mL LB Broth with 35uL ampicillin.

 

Miniprep to Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit):

 

• Transfer some of the 5mL bacterial culture into a microcentrifuge tube. Pellet by centrifugation at 13,500 rpm for 1 min. Repeat till all the culture has been pelleted.

• Resuspend the pellet in 250uL Buffer P1.

• Add 250uL Buffer P2 (Lysis Buffer). Mix thoroughly by inverting the tube 4-6 times. Do not allow prolonged lysis.

• Immediately add 350uL Buffer N3 (Neutralization Buffer). Mix immediately by inverting the tube.

• Centrifuge for 10 minutes at 13,500 rpm.

• Apply the resulting supernatant to the QIAprep spin column by pipetting. Centrifuge for 1 minute at 13,500 rpm.

• Wash the QIAprep spin column by applying 0.75mL Buffer PE. Centrifuge for 1 minute at 13,500 rpm.

• Centrifuge for an additional 1 minute to remove residual ethanol.

• Place the QIAprep spin column in microcentrifuge tube. Elute DNA by adding 50uL warm H₂O.

• Centrifuge for 1 minute at 13,500 rpm.

 

Midiprep for Large Volumes of Culture (QIAGEN Plasmid Midi Kit):

Refer to the QIAGEN website for the protocol.

PCR Amplification of Gene from Bacterial Genome/Standard Biobrick Parts

37.5μL   ddH2O

5.0μL     10x PCR Buffer

2.5μL     dNTPs

1.0μL     Forward Primer (Prefix)

1.0μL     Reverse Primer (Suffix)

1.0μL     Template DNA

1.0 μL    RTaq DNA Polymerase

50.0μL Total

 

Note:
- For sequential PCR reactions: After the first PCR is complete, perform PCR clean-up. Then use the product to conduct the second PCR reaction, after which gel purification needs to be carried out.

- The volume of DNA used in the second PCR reaction depends on the concentration of the DNA after the PCR Clean-up.

 

PCR Clean-Up – Qiagen QIAquick PCR Purification:

 

• Add 5 volumes of Buffer PB to 1 volume of PCR mix.

• Place this mix within the QIAquick column. Centrifuge at 13,500 rpm for 1 minute.

• Discard flow through and centrifuge again to allow all the sample to pass through.

• Wash with 750uL Buffer PE. Centrifuge at 13,500 rpm for 1 minute.

• Discard flow through and centrifuge again to remove all residual buffer.

• Place the column in a micro centrifuge tube.

• Apply 50uL of pre-warmed distilled H2O to the column. Stand for at least 2 minutes.

• Centrifuge at 13,500 rpm for 1 minute.

 

Colony PCR
 

14.52μL ddH2O

2.0μL     10x PCR Buffer

1.6μL     dNTPs

0.4μL     Forward Primer (Prefix)

0.4μL     Reverse Primer (Suffix)

1.0μL     Template DNA

0.08μL   RTaq DNA Polymerase

20.0μL Total

 

Notes:

- The mixture was pipetted into PCR tubes

- All materials were kept on ice

- Colonies will inoculated into 5uL broth prior to PCR

- Prefix and Suffix were used as Forward and Reverse Primers respectively while amplifying standard biobrick parts

- PCR Reaction: 95°C - 10 min

                           95°C - 30 sec

                           57°C - 30 sec (appropriate annealing temperature for prefix and suffix)

                           72°C - 30 sec

                          (28 cycles all together)

                           72°C - 5 min

 

Agarose Gel Electrophoresis

• Prepare a 2% or 1% Agarose Gel (amount in grams depending on volume of TAE buffer used). Add 0.1% Ethidium Bromide of the total volume.

• Place the gel in the Electrophoresis Apparatus with the wells facing the Negative Electrode.

• Fill the apparatus with 1% TAE Buffer to fully submerge the wells.

• Load 5µL of 1kb Ladder for each Run

• Add 0.1% of 10% Loading Dye to the respective volume of sample. Mix well and spin down.

• Pipette the samples into the wells and run at 106 Volts.

Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)

• Exercise the DNA fragment from the Agarose Gel using a scalpel. Minimize the extra peripheral gel slice.

• Weigh the gel slice (0.1g = 100uL) and add 3 Volumes of Buffer QG to every 1 Volume of Gel.

• Incubate in 50°C water bath for 10 minutes to completely dissolve the gel slice.

• Add 1 Volume of Isopropanol to the sample. Mix well

• Apply the sample to the QIAquick column. Centrifuge at 13,500 rpm for 1 minute. [Repeat till the total volume of the sample has sieved through the column]. Discard the flow through

• Apply 0.5mL Buffer QG to the QIAquick column. Centrifuge at 13,500 rpm for 1 minute.

• Discard the flow through

• Wash the column with 0.75mL Buffer PE. Centrifuge at 13,500 rpm for 1 minute.

• Discard the flow through and Centrifuge at 13,500 rpm for an additional 1 minute to eliminate any residual ethanol.

• Place the QIAquick column into a 1.5mL Eppendorf tube.

• Apply the 30uL warm H₂O to the column. Let the column stand for at least 1 minute.

• Centrifuge at 13,500 rpm for 1 minute.
   

Determining DNA Concentration Using NanoDrop Spectrophotometry

• Choose the Nucleic Acid Measurement option in the programme.

• Initialize the NanoDrop by adding 1uL clean H₂O. Clean the sensor gently with tissue.

• Set Blank by adding an additional 1uL of clean H₂O. Wipe off.

• Add 1uL of the DNA sample to be measured. Wipe off after each run.

 

DNA Digestion

__μL   ddH2O (to a total of 40uL)

4μL     10X NEBuffer

0.4μL  100X BSA

1ug     DNA Sample

__μL   1^st Restriction Enzyme

__μL   2^nd Restriction Enzyme (optional)

40μL Total

 

 Notes:

- The NEB official website should be checked for buffers suitable for each restriction enzyme. Results can vary depending on double or single digestion.

- Incubate the digestion sample at 37°C for 3 hours (digestion time can also vary depending on enzyme). Prolonged digestion may lead to Star Activity otherwise.

- The volume of DNA must be calculated from its concentration. In restriction digestion test, the minimum volume that is equals 1ug DNA can be utilized. However, for purification, a much greater volume of DNA should be used.   

- Appropriate amount of enzyme is derived from its concentration and the fact that 5 units of enzyme digest 1ug DNA.

 

Dephosphorylation of 5' Ends of Vector Backbone

- Add 0.5µL (0.5 units per 1 ug DNA) of Calf Intestinal Alkaline Phosphatase (CIAP) to the digested sample

immediately after digestion.

- Incubate at 37°C for 30 minutes

 

Vector-Insert Ligation

__μL  Autoclaved ddH2O (to a total of 20uL)

2μL    T4 Ligase Buffer

1μL    T4 DNA Ligase

__μL  Vector DNA

__μL   Insert DNA

20μL  Total

 

Notes:

- Insert and Vector must be in a 3:1 ratio. The amount of each depends on their concentration (ng/uL) and legnth (bp).

- Incubate at room temperature for 1 hour. 

 

PCR Deletion (Site-Directed Mutagenesis) Reaction

Note: Keep everything on ice and add all volumes in a PCR tube.

? µL ddH2O (? = whatever volume needed to bring the total volume up to 50µL)

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

? uL forward primer = 125ng fwd primer ÷ fwd primer concentration (ng/µL)

? uL reverse primer = 125ng rvs primer ÷ rvs primer concentration (ng/µL)

? µL dsDNA= 20ng insert ÷ insert concentration (ng/µL)

1.0μL PfuUltra high-fidelity DNA polymerase

50.0μL Total
 

- Volumes of diluted primer based on calculations for our ng/µL concentrations

95°C for 2min

95°C for 30sec (18 times)

55°C for 30sec

72°C for 1 min/kb

 

1min/kb corresponds to: 3.20min (RFP), 3.50min (VioA), 5.40min (VioB), 3.00min (VioE)