Experiments
the metdods we used most during our laboratory work were New England BioLabs HiFi DNA Assembly Cloning Kit and tdeir instructions for restriction digest and DNA-ligation.
HiFi DNA Assembly Cloning Kit.
Set up tde following reaction on ice:
| |
2-3 Fragment |
4–6 Fragment |
Positive control |
| Recommended DNA ratio |
vector:insert = 1:2 |
vector:insert = 1:1 |
|
| Total Amount of Fragments |
0,03–0,2 pmols X μl |
0,2–0,5 pmols X μl |
10 μl |
| NEBuilder HiFi DNA Assembly Master Mix |
10 μl |
10 μl |
10 μl |
| Deionized H2O |
10-X μl |
10-X μl |
0 |
| Total Volume |
20 μl |
20 μl |
20 μl |
If 2-3 fragments: Incubate sample at 50°C for 15 min.
If 4-6 fragments: Incubate at 50°C for 60 min.
Store sample on ice.
Add 2 μl of assembled product to tde competent cells. Mix gently by pipetting up and down.
Incubate on ice for 30 min.
Heat shock at 42°C for 30 sec.
Incubate on ice for 2 min.
Add 950 μl of room-temperature SOC media to tde sample.
Incubate on shake at 37°C for 60 min at 250 rpm.
Warm selection plates to 37°C.
Spread approximately 100 μl of tde cells onto tde plate and incubate at 37°C overnight.
Don’t forget positive and negative control for plates.
NEBs restriction digest and T4 DNA Ligase
Following instruction are recommended when using tde enzymes EcoRI-HF and SpeI.
| PROTOCOL FOR RESTRICTION DIGEST |
|
| Restriction Enzymes |
1 μl of each. Total Volume = 2 μl. |
| DNA |
1 μg |
| 10X NEBuffer – Cutsmart 100 |
5 μl (1X) |
| Total Rxn Volume |
50 μl |
| Incubation Temperature |
37°C |
| Incubation Time |
5-15 min |
Inactivate enzymes for 20 min at 80°C.
Set up the following reaction on ice using a microcentrifuge tube:
| PROTOCOL FOR LIGATION |
|
| T4 DNA Ligase Reaction Buffer (10X) * |
2 μl |
| Vector DNA (4kb) |
50 ng (0,020 pmol) |
| Insert DNA (1 kb) |
37,5 ng (0,060 pmol) |
| Nuclease-free water |
To 20 μl |
| T4 DNA Ligase |
1 μl |
| Total Volume |
20 μl |
*Thaw and resuspend T4 DNA Ligase Reaction Buffer at room temperature.
Mix reaction gently by pipetting up and down and microfuge briefly.
For sticky ends: Incubate at 16°C overnight or at room temperature for 10 min.
For blunt ends or single base overhangs: Incubate at 16°C overnight or at room temperature for 2 h.
Heat inactivate at 65°C for 10 min.
Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
Other methods
Gel electrophoresis with agarose.
Make 10X TAE Buffer Stock (1 liter):
48 g Tris-base
11 ml Acetate
20 ml 0,5M sodium EDTA
969 ml H20
1,5 g agarose is mixed with 100 ml of 1X TAE buffer (20 ml 10X TAE Stock + 180 ml H20). Heat in microwave to make solution homogeneous (don’t over boil). Cool solution down and then pour the agarose into a gel tray with the well comb in place. Mix samples with loading dye. Load samples and ladder to wells. Run at 90V for 80 min.
Agar for petri dishes and bacteria culture.
5 g peptone
10 g peptone from casein
10 g NaCl
12 g agar-agar
1 liter sterile H20
Autoclave before use on plates. Store at room temperature.
NEBs protocol for PCR to amplify our DNA.
Total volume of reaction is 25 μl.
1,25 μl 10μM Forward Primer
1,25 μl 10μM Reversed Primer
Variable concentration of Template DNA (<1000 ng)
12,5 μl Q5 High-Fidelity 2X Master Mix
Add to 25 μl Nuclease-Free Water
LB-medium.
10 g tryptone
5 g yeast extract
10 g NaCl
950 ml deionized water.
Autoclave before use. Store at room temperature.
