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Revision as of 11:09, 31 October 2017
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:
- Thaw competent cells on ice
- Pipette 50 micto liters of competent cells into 2ml tube
- Pipette 1 micro litre of resuspended DNA into 2ml tube
- Pipette 1 micro litre of control DNA into 2ml tube
- Close 2ml tubes, incubate on ice for 30min
- Heat shock tubes at 42C for 1 min
- Incubate on ice for 5min
Chemical Transformation:
- Pipette 200microlitre SOC media to each transformation
- Incubate at 37C for 2 hours, shaker or rotor recommended
- Pipette each transformation on 2 petri plates for a 20 micro litre and 200 micro litre plating
- Incubate transformations overnight
- APick single colonies
- Count colonies for control transformation
Materials (consumables):
5µl NEB buffer 2
0.5µl EcoRI-HF
0.5µl PstI
19µl dH20
Ice
Methods:
- Add 4µl linearized plasmid backbone (25ng/µl for 100ng total)
- Add 4µl of Enzyme Master Mix
- 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:
- Add 10µl of part DNA (100ng total)
- Add 4µl of enzyme Mix
- Digest 37⁰C/30min, heat kill 80⁰C/20min
Materials (consumables):
LB broth
Bacteriological Agar
100mg/ml Chloramphenicol Solution
PREPARATION OF LB-AGAR PLATE (500ml)
- Add 12.5g of LB broth powder into an appropriate bottle
- Add 7.5g of Agar
- Autoclave
- Pour hot/ warm (melted) media at 20ml per petri dish
PREPARATION OF LB-AMP PLATE (500ml)
- Add 12.5g of LB broth powder into an appropriate bottle 2
- Add 7.5g of Agar
- Autoclave
- Allow media to cool till it can be felt at the back of the palm without burning (but still not solidified).
- Add 500µl of 100mg/ml Chloramphenicol solution
- Swirl to mix the antibiotic uniformly in the media
- Pour the melted media at 20ml per petri dish
Run gel:
- Add 5µl of PCR solution and 1µl 10x loading dye
- Load 6µl of DNA ladder alongside and all samples (Do not forget to add dye to ladder too) - NEB 1kb ladder used
- Run gel at 100V for 45 min
- 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:
- Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube.
- 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.
- Add 350 µl of cold Neutralization Buffer (Yellow) and mix thoroughly
- Place the column into a Collection Tube and centrifuge for 15 seconds.
- Discard the flow-through and place the column back into the same Collection Tube.
- Add 200 µl of Endo-Wash Buffer to the column
- Add 400 µl of Zyppy™ Wash Buffer to the column
- 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.
- . Centrifuge for 30 seconds to elute the plasmid DNA.
