Team:AshesiGhana/Protocols

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This page shows general protocols given by the suppliers, supervisors and/or published papers.

Materials
Resuspended DNA
10pg/ul Control DNA
Competent Cells
2ml Microtubes
Floating Foam Tubes
Ice $ Ice Bucket
Lab Timer 42 °C water bath
SOC Media
37C Incubator
Pertri plates w/ LB agar and antibiotic
Sterile Spreader
Pipettes and Tips

Preparation of chemical competent cells:

  1. Thaw competent cells on ice
  2. Pipette 50 micto liters of competent cells into 2ml tube
  3. Pipette 1 micro litre of resuspended DNA into 2ml tube
  4. Pipette 1 micro litre of control DNA into 2ml tube
  5. Close 2ml tubes, incubate on ice for 30min
  6. Heat shock tubes at 42C for 1 min
  7. Incubate on ice for 5min

Chemical Transformation:

  1. Pipette 200microlitre SOC media to each transformation
  2. Incubate at 37C for 2 hours, shaker or rotor recommended
  3. Pipette each transformation on 2 petri plates for a 20 micro litre and 200 micro litre plating
  4. Incubate transformations overnight
  5. APick single colonies
  6. Count colonies for control transformation

Materials (consumables):
5µl NEB buffer 2
0.5µl EcoRI-HF
0.5µl PstI
19µl dH20
Ice

Methods:

  1. Add 4µl linearized plasmid backbone (25ng/µl for 100ng total)
  2. Add 4µl of Enzyme Master Mix
  3. MDigest 37⁰C/30min, heat kill 80⁰C/20min.

Materials (consumables):
5µl NEB Buffer 2
0.5µl EcoRI-HF
0.5µL SpeI
DNA template
19µl dH2O

Methods:

  1. Add 10µl of part DNA (100ng total)
  2. Add 4µl of enzyme Mix
  3. Digest 37⁰C/30min, heat kill 80⁰C/20min

Materials (consumables):
LB broth
Bacteriological Agar
100mg/ml Chloramphenicol Solution

PREPARATION OF LB-AGAR PLATE (500ml)

  1. Add 12.5g of LB broth powder into an appropriate bottle
  2. Add 7.5g of Agar
  3. Autoclave
  4. Pour hot/ warm (melted) media at 20ml per petri dish

PREPARATION OF LB-AMP PLATE (500ml)
  1. Add 12.5g of LB broth powder into an appropriate bottle 2
  2. Add 7.5g of Agar
  3. Autoclave
  4. Allow media to cool till it can be felt at the back of the palm without burning (but still not solidified).
  5. Add 500µl of 100mg/ml Chloramphenicol solution
  6. Swirl to mix the antibiotic uniformly in the media
  7. Pour the melted media at 20ml per petri dish

Run gel:

  1. Add 5µl of PCR solution and 1µl 10x loading dye
  2. Load 6µl of DNA ladder alongside and all samples (Do not forget to add dye to ladder too) - NEB 1kb ladder used
  3. Run gel at 100V for 45 min
  4. Visualise gel on a transilluminator (SYBR Safe binds DNA and fluoresces under UV light)

Materials (consumables):
7X Lysis Buffer*1 (Blue)
Neutralization Buffer*2 (Yellow)
Endo-Wash Buffer
Zyppy™ Elution Buffer
Collection Tubes

Methods:

  1. Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube.
  2. Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes.
  3. Add 350 µl of cold Neutralization Buffer (Yellow) and mix thoroughly
  4. Place the column into a Collection Tube and centrifuge for 15 seconds.
  5. Discard the flow-through and place the column back into the same Collection Tube.
  6. Add 200 µl of Endo-Wash Buffer to the column
  7. Add 400 µl of Zyppy™ Wash Buffer to the column
  8. Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer2 directly to the column matrix and let stand for one minute at room temperature.
  9. . Centrifuge for 30 seconds to elute the plasmid DNA.