Protocols
Preparation of chemically competent DH5alpha E. coli cells
Material:
- LB media
- TSS buffer
- DH5alpha E. coli cells (o/n colonies on agar plates)
Method:
- Pick one colony of the plate and transfer into 5 mL of LB media. Grow the culture over night for 16-18 hours at 37 deg. celsius.
- Transfer 1 mL of the overnight culture into a shaking flask with 99 mL of LB media. Measure optical density (OD) at 600 nm and incubate culture at 37 deg. celsius (shaking) to an OD of 0,5.
- Divide the 100 mL into two 50 mL tubes and incubate 10 min on ice.
- Spin the tubes at 3000 rpm for 10 minutes at 4 deg. celsius.
- Resuspend the pellet of competent cells with 10 % TSS buffer (5 mL).
- Aliquot 100 µL of the cell solution into 1.5 mL microtubes (all steps on ice!).
- Store the competent cells at -80 deg. celsius.
Heat-shock Transformation of E. coli cells
Material:
- SOC media
- Agar plates with appropriate antibiotic
Method:
- Thaw chemically competent cells on ice.
- Transfer 50 µL of the cells into a 1.5 mL microtube, add 1 µL of the desired DNA and incubate on ice for 30 minutes.
- Place the tube in a 42 deg. celsius water bath for 60 seconds.
- After heat shock leave cells on ice for 5 minutes.
- Add 950 µL of SOC media and shake cells for 2 hours at 37 deg. celsius.
- Pipet 100 µL of the cells onto an appropriate plate and spread them using sterile glass beads. Incubate overnight at 37 deg. Celsius and hope for colonies in the morning to prove a successful transformation.
Preparation of LB media
Material:
- Tryptone
- NaCl
- Yeast extract
Method:
- Fill a container (bottle) to about 60/70 % its volume with destilled water.
- Add 10 g/L Tryptone, 10 g/L NaCl and 5 g/L of yeast extract.
- Stir properly and fill up the remaining volume with distilled water.
- Treat LB media by autoclave.
Preparation of LB agar
Material:
- Tryptone
- NaCl
- Yeast extract
- Agar
Method:
- Fill a container (bottle) to about 60/70 % its volume with destilled water.
- Add 10 g/L Tryptone, 10 g/L NaCl, 5 g/L of yeast extract and 20 g/L agar.
- Stir properly and fill up the remaining volume with distilled water.
- Treat by autoclave.
Glycerol stocks – storage of bacterial strains
Material:
Method:
- Mix 700 µL of overnight culture with 300 µL glycerol.
- Store at -80 deg. Celsius.
Polymerase chain reaction (PCR)
Material:
- Primer
- Template DNA
- NEB Q5® High-Fidelity 2X Master Mix (dNTPs + Polymerase)
- distilled water
- PCR-Cycler
Method:
- All steps have to be performed on ice.
- 50 µL approach (mix well):
| components | volume | concentration |
|---|
| Q5 Master Mix | 25 µL | 1x |
| 10 µM fw primer | 2,5 µL | 0,5 µM |
| 10 µM rv primer | 2,5 µL | 0,5 µM |
| template DNA | | <1000 ng |
| water | remaining volume to 50 µL |
- PCR-cycler conditions:
| step | cycles | temperature | time |
| Denaturation | 1 | 98°C | 30 sec |
| Annealing | 25-35 | 98°C | 5-10 sec |
| Elongation | | 72°C | 20-30sec/kb |
| final extension | 1 | 72°C | 2 min |
| hold | | 4-10°C | |
Determination of DNA concentration
DNA concentration is determined by using a Nanodrop (). The absorbance at 260 nm is converted to concentration using the Lambert – Beer Equation by the program. The purity of the sample is confirmed by the 260/280 ratio (contamination with proteins) and the 260/230 ratio (presence of co-purified contaminants). For pure DNA the 260/280 ratio should be around 1.8 and the 260/280 ratio should be around 1.8 – 2.2. (ND-1000-v3.4-users-manual, Thermo Scientific)
Mini-Prep (based on Fast-n-Easy Plasmid Mini-Prep Kit Jena Bioscience)
Material:
- Lysis buffer
- Neutralization buffer
- Column-Activation buffer
- Wash buffer
- Elution buffer
- Binding column
Method:
- Harvest the over-night culture by centrifugation (3000 g for 10 minutes.)
- Activate the Binding Column by adding 100 µl of Activation buffer and centrifugation at 10000 g for 30 seconds.
- For cell lysis resuspend the cell pellet in 300 µl Lysis buffer (pipetting or vortexing).
- Add 300 µl of Neutralization buffer and mix by inverting the tube (4 – 6 times).
- Centrifuge at 10000 g for 5 minutes.
The colour of the supernatant should be yellow.
- Transfer the supernatant into the Binding Column and centrifuge at 10000 g for 30 seconds. Discard the flow-through.
- Add 500 µl Washing buffer to the column and centrifuge at 10000 g for 30 seconds. Discard the flow-through.
- Place the Binding Column into a clean microtube an add 30 µl Elution buffer.
- Incubate for 1 minute and centrifuge at 10000 g for 1 minute to elute DNA.
- The eluted DNA could be used directly or should be stored at -20°C.
Restriction digest
Materials:
- Plasmid DNA
- Restriction Enzymes
- Restriction buffer
- H2O
- Ice
Method:
- All steps must be performed on ice.
- For a 20 µl double digest approach following amount are needed:
| components | volume | concentration |
| restriction buffer (10X) | 2 µL | 1X |
| restriction enzyme 1 | 1 µL | |
| restriction enzyme 2 | 1 µL | |
| plasmid DNA | x µL | 1 µg total |
| H2O | add to 20 µL | |
- Digest the approach for 1h at 37 °C.
- To stop the reaction, incubate the reaction for 20 minutes at 80 °C.
Gel-Extraction (based on Gel DNA Recovery Kit from Zymo Research)
Material:
- Extraction buffer (ADB buffer)<(li>
- Washing buffer
- Elution buffer
- Spin column
Method:
- Cut out the area of the gel containing the DNA fragment of interest.
- Add ADB buffer (3 volumes to 1 volume gel).
- Dissolve the gel by incubation at 37 – 55 °C for 5 – 10 minutes.
- Transfer the solution to a spin column and centrifuge for 30 seconds. Discard the flow-through.
- Add 200 µl Washing buffer and centrifuge for 30 seconds. Discard the flow-through.
- Repeat the wash step.
- Place the column in a clean microtube and add more than 6 µl Elution buffer. Centrifuge for 1 minute to elute DNA.
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