1) Anammox is originated from Bacteria, which implied that using cell extracts from E.coli would be unlikely to produce erroneous result.
2) E.coli cell free Expression System is the quickest and most efficient way of CFE.
| Component | Volume |
|---|---|
| Insert or Vector DNA (100 pg/μL – 1 ng/μL in TE) | 0.5 μL |
| 10 μM Forward Primer | |
| 10 μM Forward Primer | 2.5 μL |
| 10 μM Reverse Primer | 2.5 μL |
| 10 mM dNTPs | 1 μL |
| 10 mM dNTPs | 1 μL |
| 5X Phusion HF Buffer | 10 μL |
| Phusion® DNA Polymerase (2 U/ μL) | 0.5 μL |
| Nuclease-free Water | 33 μL |
| Total | 50 μL |
a. Operation of Thermocycle
| Step | Temperature | Duration | Number of cycles |
|---|---|---|---|
| Initial denaturation | 98°C | 30 seconds | 1 cycle |
| 98°C | 10 seconds | ||
| Amplification | Primer Tm(60°C to 70°C) | 20 seconds | 25–30 cycles |
| 72°C | 30 seconds per kb | ||
| Final extension | 72°C | 5 minutes | 1 cycle |
| Hold | 4°C | - | 1 cycle |
b. Purification
1. Thaw GA 1-Step Master Mix (2X) on ice.
2. In PCR tubes, prepare DNA fragments in nuclease-free using the following formula.
[pmol DNA = [ng DNA/(660 x # of bases)] x 1000]
3. Vigorously vortex the master mix for 15 seconds immediately before use after it is thawed.
4. In a tube on ice, combine 5 μL of DNA fragments and 5 μL of GA 1-Step Master Mix (2X). Mix the reaction by pipetting.
5. For the positive control, combine 5 μL of the Positive Control (2X) and 5 μL of GA 1-Step Master Mix (2X) in a tube on ice. Mix the reaction by pipetting.
6. Vortex and spin down all reactions.
7. Incubate the reactions at 50°C for 1 hour.
8. After the incubation is complete, store the reactions at −20°C or dilute reactions for the downstream application, CFE.
9. Analyze the assembly reaction by performing gel electrophoresis with 5–10 μL of the reaction on an 0.8–2% agarose gel.
1. For each sample, add the following reagents to the appropriate reaction vessel:
| Reagent | Amount |
|---|---|
| E. coli slyD- Extract | 20 µl |
| 2.5X IVPS E. coli Reaction Buffer (-A.A.) | 20 µl |
| 50 mM Amino Acids (-Met) | 1.25 µl |
| 75 mM Methionine* | 1 µl |
| T7 Enzyme Mix | 1 µl |
| DNA Template | 1 µg |
| DNase/RNase-free Distilled Water | To a final volume of 50 µl |
2. Close the tube and incubate sample in a standard shaking incubator (300 rpm) at 30°C for 30 minutes. If the protein you are synthesizing is known to be soluble, you may incubate the sample at 37°C.
3. During the 30 minute incubation, prepare the Feed Buffer. For each sample, add the following reagents to a sterile, RNase-free microcentrifuge tube. For multiple samples, you may scale up the volume of reagents used accordingly and prepare one master mix.
| Reagent | Amount |
|---|---|
| 2X IVPS Feed Buffer | 25 µl |
| 50 mM Amino Acids (-Met) | 1.25 µl |
| 75 mM Methionine* | 1 µl |
| DNase/RNase-free Distilled Water | To final volume of 50 µl |
4. After 30 minutes of incubation (from Step 2 above), add 50 µl of the Feed Buffer to the sample (total volume = 100 µl).
5. Cap the tube and return the sample to the shaking incubator (300 rpm). Incubate for 3-6 hours at 30-37°C as appropriate.
6. Place the reaction on ice and proceed to Analyzing Samples, or store the sample at -20°C for future processing or analysis.
1) Combine the following reagents:
| Reagent | Amount |
|---|---|
| 0.5 M Tris-HCl, pH 6.8 | 2.5 ml |
| Glycerol (100%) | 2 ml |
| β-mercaptoethanol | 0.4 ml |
| Bromophenol blue | 0.02 g |
| SDS | 0.4 g |
2) Bring the volume to 20 ml with sterile water.
3) Aliquot and freeze at -20°C until needed.
a. Acetone Precipitation
1. Add 5 µl of the protein reaction product from Step 6, previous page, to 20 µl of acetone. Mix well.
2. Centrifuge for 5 minutes at room temperature in a microcentrifuge at 12,000 rpm.
3. Carefully remove the supernatant, taking care not to disturb the protein pellet.
4. Resuspend pellet in 20 µl of 1X SDS-PAGE sample buffer.
5. Heat at 70-80°C for 10-15 minutes and centrifuge briefly. Proceed to Polyacrylamide Gel Electrophoresis on the next step.
b. Polyacylamide Gel Electrophoresis
1. Load 5-10 µl of the sample from Step 6, previous page on an SDS-PAGE gel and electrophorese at 120V. You may save your sample by storing at -20°C, if desired.
2. Stain gel with Coomassie blue stain or other stain.
a. Total Count Determination
1. Mix and spot 5 µl of each radiolabeled reaction from Step 6 above on a glass microfiber filter (Type GF/C; Whatman).
2. Set aside and let dry. Do not wash or TCA precipitate these filters.
b. TCA Precipitation
1. Mix and spot 5 µl of each radiolabeled reaction from Step 6, page 21 on a separate set of individual glass fiber (GF/C) filters and allow to air dry for approximately 5-10 seconds.
2. Place filter in a beaker and wash once with cold 10% TCA for 10 minutes at room temperature while shaking gently (use approximately 10-20 ml per filter).
3. Wash with 5% TCA for 5 minutes at room temperature while shaking gently. Repeat wash.
4. Rinse filters with methanol to facilitate drying.
5. Allow filters to dry, place in scintillation vials, and add scintillation fluid. Count samples in a scintillation counter.
6. Proceed to Calculating Protein Yield on the next step.
c. Protein Yield Calculation
