Team:Hamburg/Experiments
Experiments
On this page we only display our protocols. If you are looking for detailed information on important experiments, visit our Results page and the registry entries of our Parts. If you are looking for detailed information on any experiment not mentioned in Results, visit our Notebook.
- 0.5 g Agarose
- 50 mL TAE-buffer (gel room)
- Heat in microwave until agarose is dissolved
- Transfer into gel slide, tip pipet into EtBr, then into gel
- Cool for 30 minutes
34 mg/mL preparation of Choramphenicol Stock (3 in EtOH)
- dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol
- filter through a 0.22 μL filter to sterilize
- use at 1:1000 dilution in LB or LB-agar
- mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics
- store at -20 °C
-
Ca/glycerol buffer: 60mM CaCl2, 10mM PIPES, 150mL glycerol, Fill water up to 1L, pH=7.0
Filter sterilization. Avoid autoclaving.
- Grow the Cells at 37°C till OD=0.2-0.4
- Chill the culture on ice for 5min.
- Cllect cells by centrifugation, 6000rpm 10min 4°C
- Gently resuspend the cells from 500ml LB in 40ml cold Ca/glycerol buffer on ice.
- Incubate the cells on ice for 5~30min.
- Repeat step 3 and 4.
- Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The longer, the better)
- Collect cells by centrifugation, 6000rpm 10min 4°C
- Resuspend cells in 6ml Ca/glycerol buffer.
- Aliquote 150µl/tube. Freeze in liquid N2 and store at -80°C.
PURIFICATION PROTOCOLS
Note
- Read IMPORTANT NOTES on p.3 before starting.
- All purification steps ahould be carried out at room temperature.
- All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).
Protocol A. DNA purification using centrifuge
| Step | Procedure |
|---|---|
| 1 | Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow. |
|
2
for DNA ≥500 bp |
Optional: if the DNA fragment is ≥ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
Note. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower. |
| 3 |
Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.
Note. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use! |
| 4 |
Add a 700 µL of Wash Buffer (diluted with the ethanol as described on p.3) to the GeneJET purification column. Centrifuge for 30-60 s.
Discard the flow-through and place the purification column back into the collection tube. |
| 5 |
Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
Note. This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions. |
| 6 |
Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).
Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min. Note.
|
| 7 | Discard the GeneJET purification column and store the purified DNA at -20 °C. |
Growth of Bacterial Cultures
- Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask with a volume of least 4 times the culture volume.
- Havest the bacterial culture by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room temperature. Decant the supernetant and remove all remaining medium.
For high-copy-number plasmids (see Table 1), do not process more than 5 mL of bacterial culture. If more than 5 mL of such a culture are processed, the GeneJET spin column capacity (20 μL of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.
For low-copy-number plasmids (see Table 1), it may be necessary to process larger volumes of bacterial culture (up to 10 mL) to recover a sufficient quantity of DNA.
Table 1. Copy numbers of various vectors
|
High-copy
300-700 copies per cell |
Low-copy
10-50 copies per cell |
Very low-copy
up to 5 copies per cell |
|
pUC vectors
pBluescript vectors pGEM vectors pTZ vectors pJET vectors |
pBR322 and derivatives
pACYC and derivatives |
pSC101 and derivatives |
PURIFICATION PROTOCOLS
Note
- Read IMPORTANT NOTES on p.3 before starting.
- All purification steps should be carried out at room temperature.
- All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).
Use 1-5 mL of E. coli culture in LB media for purification of higt-copy plasmids.
For low-copy plasmids use up to 10 mL of culture.
Protocol A. Plasmid DNA purification using centrifuges
| Step | Procedure |
|---|---|
| 1 |
Resuspend the pelleted cells in 250 μL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
Note. Ensure RNase A has been added to the Resuspension Solution (as describe on p.3) |
| 2 |
Add
250 μL of the Lysis Solution
and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA. |
| 3 |
Add
350 μL of the Neutralization Solution
and mix immediately and thoroughly by inverting the tube 4-6 times.
Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy. |
| 4 | Centrifuge for 5 min to pellet cell debris and chromosomal DNA. |
| 5 |
Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white prepipitate.
Note. Close the bag with GeneJET Spin Columns tightly after each use! |
| 6 |
Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note. Do not add bleach to the flow-through, see p.8 for Safety Information. |
|
7
for EndA+ strains only |
Optional: use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used.
Wash the GeneJET spin column by adding 500 μL of Wash Solution I (#R1611, diluted) |
| 8 | Add 500 μL of Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centriifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. |
| 9 | Repeat the wash procedure (step 8) using 500 μL of the Wash Solution. |
| 10 | Discard the flow-trough and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. |
| 11 |
Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add
50 μL of the Elution Buffer
to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20% For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70 °C before applying to silica membrane. |
| 12 | Discard the column and store the purified plasmid DNA at -20 °C. |
from liquid culture
- 500 μL (50% Glycerol, 50% H2O)
- 500 μL Cells
- Store at -80 °C
- Primer concentration: 100 μM → Concentration needed: 5 μM (dilute at 1:20)
- take 50 nm of sample mix with 5 μL of (5 μM primer, only forward)
Choose enzymes and DNA amount:
|
Fragment |
Enzymes |
DNA amount |
|
Vector |
EcoRI, PstI, FastAP |
1000 ng |
|
Single insert (e.g. PCR product) |
EcoRI, PstI |
500 ng |
|
Assembly insert 1 |
EcoRI, SpeI |
500 ng |
|
Assembly insert 2 |
XbaI, PstI |
500 ng |
Set up digestion reactions with DNA volume according to needed amount, 1 µL of each chosen enzyme, NEBuffer 2.1 to match one tenth of the total reaction volume, and H2O to fill up to desired reaction volume.
Incubate digestion at 37°C for an hour prior to heat inactivation at 80°C for 15 minutes. Run a 1% agarose gel with all samples and extract desired bands using GeneJET Gel Extraction Kit (ThermoFisher).
Set up ligations with 10-fold molar excess of inserts over vector, with total DNA per ligation to match 200 ng. Add 1 µL T4 DNA Ligase and T4 ligase buffer (ThermoFisher) to match one tenth of the total reaction volume. Add H2O to fill up to desired reaction volume. Incubate ligation at 37°C for 1 h prior to heat inactivation at 80°C for 15 minutes. Transform competent DH5α with all the DNA according to Zhang’s transformation protocol.
Zhang Gong
Thaw competent DH5α at 4°C on ice for 5 minutes. Inoculate cells with up to 200 ng DNA, and incubate cells at 4°C on ice for 1 h. Heat shock cells at 42°C for 45 s, and immediately put them back at 4°C on ice for 5 min. Add 850 µL LB medium and incubate at 37°C, 900 RPM on the thermomix for 1h. Spin down cells, discard 800 µL supernatant and resuspend cells in the remaining medium. Bring cells onto LB-Agar plates with appropriate antibiotics and incubate at 37°C overnight.
Funding
