Team:Bulgaria/PROTOCOLS
Miniprep Plasmid Purification
1. Resuspend the pelleted cells in 250 uL 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.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. 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 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. 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 precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL 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.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.
PureLink® Quick Gel Extraction and PCR Purification Combo Kit
Purifying PCR fragments or reaction clean-up
1. Combine. Add 4 volumes of Binding Buffer (B2) with isopropanol to 1 volume of a PCR sample (50–100 uL). Mix well.
2. Load. Pipet the sample into a PureLink® Clean-up Spin Column in a Wash Tube. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.
3. Wash. Re-insert the column into the Wash Tube and add 650 uL Wash Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute.
4. Remove ethanol. Discard the flow-through and place the column in the same Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes.
5. Elute. Place the column into a clean 1.5-mL Elution Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum speed for 1 minute.
6. Store. The elution tube contains the purified PCR product. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.
Purifying DNA from Gels Using a Centrifuge
1. Excise. Use a clean, sharp razor blade to excise a minimal area of gel containing the DNA fragment of interest.
2. Weigh. Using a scale sensitive to 0.001 g, weigh the gel slice containing the DNA fragment.
3. Solubilize. Add Gel Solubilization Buffer (L3) to the excised gel in a tube as indicated in the table. Incubate the tube at 50°C for 10 minutes (or longer for large gel slices and high concentration gels), and invert the tube every 3 minutes. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. Add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
| Gel | Tube | Buffer L3 Volume: |
|---|---|---|
| ≤ 2% agarose | 1.7-mL polypropylene | 3:1 |
| > 2% agarose | 5-mL polypropylene | 6:1 |
4. Load. Pipet the dissolved gel piece onto a column inside a Wash Tube. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Note: The column reservoir capacity is 850 uL. Use 1 column per 400 mg of agarose gel.
5. Wash. Add 500 uL Wash Buffer (W1) containing ethanol to the column. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes. Discard the flow-through.
6. Elute. Place the column into a Recovery Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the tube for 1 minute at room temperature. Centrifuge the tube at >12,000 × g for 1 minute.
7. Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.
Oligo annealing and ligation protocol for gRNAs
1. Add 1,5 ul Oligo1 (100 uM stock) and 1,5 ul Oligo2 (100 ul stock) to 500 ul oligo annealing buffer.
2. Incubate for 5' at 95°C.
3. Cool down in 500-800 ml boiling water to RT.
4. Use 4 ul or 8 ul for ligation with 200 ng linearized vector.
5. Store at -20°C.
Ligation
4 ul oligos (16 ng) or 8 ul
200 ng plasmid
2 ul 50% PEG 4000
2 ul 10x T4 ligase buffer
1 ul T4 ligase 5U/ul
Oligo annealing buffer
1 mM Tris pH 7.5
50 mM NaCl
1 mM EDTA
Ligation
1. Add the following reagents to a PCR tube: Reagent Amount 5X Ligase Reaction Buffer Vector DNA 50-100 ng Insert DNA (1:1 or 3:1 ratio) ExpressLink™ T4 DNA Ligase 5 units (in 1 uL) autoclaved distilled water to 20 uL.
2. Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube.
3. Incubate at room temperature for 5 - 15 minutes.
4. Use 2 uL of the ligation reaction to transform 100 uL of electrocompetent cells.
Glycerol Stocks
1. Pick a single colony of the clone off of a plate and grow an overnight in the appropriate selectable liquid medium (e.g., LB amp).
2. Make a label (clone ID # and date) for the construct.
3. Add 0.5ml of the o/n culture to 0.5ml of 80% sterile glycerol in the sterile screw cap microcentrifuge tube.
4. Screw a lid onto the tube and write the clone ID # on the lid of the tube.
5. Vortex.
6. Freeze the glycerol stock at –80°C.
7. To streak out from a glycerol stock:
1. Determine the location (-80°C tower #, box #, row #) of the construct.
2. Take the tube to the place that you intend to streak the clone out.
3. Flame a metal inoculating loop until it is red hot.
4. Scrape off a portion from the top of the frozen glycerol stock and streak it onto your plate.
5. Return the construct to the –80°C.
gBlock Fragment Amplification
1. Phusion® DNA Polymerase amplification reaction
Nuclease-free
H2O Adjust to final 50 uL
5X Phusion HF
10 uL 10 mM dNTPs
1 uL 10 uM Forward Primer
2.5 uL 10 uM Reverse Primer
2.5 uL gBlocks® Gene Fragments
0.1–1.0 ng Phusion® DNA Polymerase 0.5 uL
Total volume 50 uL
2. PCR program:
Initial denaturation: 98°C, 30 seconds
Cycle start:
Denaturation 98°C, 10 seconds
Annealing 60°C, 30 seconds
Extension 72°C, 20 seconds per kb
Cycle end (25-35 cycles)
Final extension 72°C, 5 minutes
Hold 4°C
TSS Competent Cells Preparation and Transformation
This protocol of competent cell preparation follows the TSS preparation described by Chung & Miller (Chung, C. T. & Miller, R. H.; Preparation and storage of competent Escherichia coli cells Methods Enzymol, 1993, pp. 621-627). We have tested the method described below with two different E. coli strains: DH5alfa and Top10. Nevertheless, all optimizations have been made using the DH5alfa strain.
1. Resuspend the pelleted cells in 250 uL 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.2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. 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 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. 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 precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL 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.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.