Experiments
The methods we used most during our laboratory work were New England BioLabs HiFi DNA Assembly Cloning Kit and their instructions for restriction digest and DNA-ligation.
Team:Linkoping Sweden/Experiments
HiFi DNA Assembly Cloning Kit.
Set up the 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 the 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 the sample. Incubate on shake at 37°C for 60 min at 250 rpm. Warm selection plates to 37°C. Spread approximately 100 μl of the cells onto the 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 the 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.
Dot-blot
For this we used Immun-StarTM AP Chemiluminescent Protein Detection Systems. Here is the instruction manual.
Lysis the bacteria with B-per Proper the membrane by soaking it with MeOH for some seconds and then put the membrane into the transfer buffer for 5 min. The membrane is then put on a filter paper which lays upon a sponge to create a transfersandwich. Transfer of proteins onto membranes and let it soak in. (if the membrane is dry, use MeOH to soak it) Soak the membrane with BSA in a bowl for 2 hours and place it on a rotating table at room temperature. Replace the BSA Blocking solution with primary antibodies. let it sit in the refrigerator overnight. Wash with deionised water. Do 3 washes with 30 ml TBST. Each wash will be incubated on a rotating table for 5 minutes. Apply secondary antibodies and let it sit 30 minutes on a rotating table for. Repeat the step with 3 washes. Do 2 washes with 30 ml TBS and let it sit on a rotating table for 5 minutes in room temperature. The membrane is covered with the substrate CDP-star chemiluminescent with enhancer. This activate the enzyme that is conjugated to the secondary antibodies. Take a picture with a CCD camera. These pictures will show the regions where the secondary antibodies have bound in. Here, the enzyme will cleave CDP-star chemiluminescent. When this happens photons will be emitted and registered by the camera.
