We are describing our research work. Below you can find the protocols we used.
Preparation restriction of components:
Ligation of cassettes into plasmids:
plasmid | cassettes | ||
---|---|---|---|
psB1C3 | 1 | 2 | 3 |
psB1A3 | 1 | 2 | 3 |
psB1K3 | 1 | 2 | 3 |
Final preparation steps
Next day
3) 3A-assembly
Legend
aim:
Is the insert DNA in the plasmid present or absent?
Much easier than to isolate, purify the vector
good to know before the start:
- Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips
- Include a no-template control and positive control in parallel.
- Thaw and keep reagents on ice
- Mix well before use.
- The longer the amplicon, the longer the extension time: Use 15 sec/kb extension.
- Use 90 sec extension for multiplexing
- Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing.
- ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2% agarose TAE gel the dye migrates with~350 bp DNA, in 1% agarose TAE gel with ~ 600 bp DNA fragments
step by step for E.coli:
- Prepare masterplate - Prepare a PCR master mix (always prepare at least 10 % more, use the excel sheet by Sophia to calculate)
- 20 μl would be good, Fabian suggested 5 μl
Mix gently, avoid bubbles.
- Aliquote the 22.0 μl of PCR master mix into each PCR tube.
- Pick colony and put the toothpick or tip one into the Masterplate and then in the PCR tube/mix.
Do not forget the negative control!
- Close tube
- Perform the PCR using Thermocycler as follow:
Initial denaturation |
1 cycle |
95°C | 60s |
---|---|---|---|
Denaturation | 30-40 cycles |
95°C | 15s |
Annealing | 30-40 cycles |
55-65°C | 15s |
Extension | 30-40 cycles |
72°C | 15-90s (15 sec per 1 kb) |
Final extension | 1 cycle |
72°C | 5 min |
- Store probes for short time on ice, for long at -20°C
- Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round).
step by step for yeast: - If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks, transfer transformants to individual tubes/wells. The amount of cells resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for ≥ 5 min at 37 °C.
If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at 37 °C.
- Prepare a PCR master mix (always prepare at least 10% more, use the excel sheet to calculate) - Aliquot 22.5 μl of PCR master mix into each PCR tube.
- Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the appropriate PCR tube.
- Close the tubes
- Perform the PCR using the following cycling profle:
Initial denaturation |
1 cycle |
95°C | 60s |
---|---|---|---|
Denaturation | 30-40 cycles |
95°C | 15s |
Annealing | 30-40 cycles |
55-65°C | 15s |
Extension | 30-40 cycles |
72°C | 15-90s |
Final extension |
1 cycle |
72°C | 5 min |
- Load probes on the agarose gel
- Store probes for short time on ice, for long at -20°C
Depending on the PCR product
If there is more than the wanted DNA band: A. Dissolving the Gel Slice |
If there iss only one DNA band: B. Processing PCR Amplifications |
---|---|
1. Following electrophoresis, excise DNA band from gel (with scalpel) and place gel slice in a 1.5 ml microcentrifuge tube. 2. Add 10 μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50 – 65 °C until gel slice is completely dissolved. |
You can just work with the rest of your PCR aliquote (when you did not use all of it for the gel electrophoresis). |
Binding of DNA
1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 ×g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
4. Heat NE-buffer to 70 °C.
Washing
5. Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
6. Repeat Step 4 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
7. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open(or off to allow evaporation of any residual ethanol.
Elution
8. Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.
9. Add 50 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
10. Discard Minicolumn and measure the concentration.
11. Store DNA at 4°C or –20°C.
– uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode
- Shorter DNA fragments migrate through the gel more quickly than longer ones
Why are we doing it ?
– to determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths)
Protocol :
- Pouring a Standard 1% Agarose Gel:
- Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.).
It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on it as the initial boil has a tendency to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close attention.
Pouring of the gel
- Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins.
- Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
- Pour the agarose into a gel tray with the well comb in place.
Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette trip.
Loading Samples and Running an Agarose Gel:
- Add loading buffer to each of your digest samples.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high percentage of glycerol, so it increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer. 2. Once solidified, place the agarose gel into the gel box (electrophoresis unit). 3. Fill gel box with 1xTAE (or TBE) until the gel is covered. 4. Carefully load a molecular weight ladder into the first lane of the gel. Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raising the pipette straight out of the buffer. 5. Carefully load your samples into the additional wells of the gel. 6. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. Note: Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always Run to Red. Note: A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage. 7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. 8. Using any device that has UV light, visualize your DNA fragments. Note: Fabian or Lena will give you a short introduction on how to work with UV light !!! Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat. Note: If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA. Note: The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel. Analyzing Your Gel: Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can interpret the bands that you get in your sample lanes to determine if the resulting DNA bands that you see are as expected or not. For more details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page. Purifying DNA from Your Gel: If you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For instructions on how to do this, visit the Gel Purification page
1.Production of cleared lysate
1. Isolation of the bacteria
1. harvest 1–5 ml (high-copy-number plasmid) or 10 ml (low-copy-number plasmid)
of bacterial culture
2. centrifugation for 5 minutes at 10,000 xg in a tabletop centrifuge
3. pour off the supernatant
4. reinsert again bacterial culture to the pellet and repeat step 2 and 3
5. blot the inverted tube on a paper towel to remove excess media
2. Resuspension of the cells
1.
add 250 μl of Cell Resuspension Solution
2.
completely resuspend the cell pellet by vortexing or pipetting
3.
it is essential to thoroughly resuspend the cells
3. Lysing
1.
add 250 μl of Cell Lysis Solution
2.
mix by inverting the tube 4 times - do not vortex
3.
incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes)
4. Splitting proteins
1.
add 10 μl of Alkaline Protease Solution
2.
mix by inverting the tube 4 times - do not vortex
3.
incubate for 5 minutes at room temperature
5. Neutralization
1.
add 350 μl of Neutralization Solution
2.
immediately mix by inverting the tube 4 times - do not vortex
6. Isolation of the plasmids
1. centrifuge the bacterial lysate at maximum speed (around 14,000 ×g) in a microcentrifuge for 10 minutes at room temperature
2. Isolation of the plasmid DNA
1. Transfer the cleared lysate (approximately 850 μl, Section 3.B, Step 6) to the prepared Spin Column by decanting. Avoid disturbing or transferring any of the white precipitate with the supernatant.
1. If the white precipitate is accidentally transferred to the Spin Column, pour the Spin Column contents back into a sterile 1.5ml microcentrifuge tube and centrifuge for another 5–10 minutes at maximum speed. Transfer the resulting supernatant into the same Spin Column that was used initially for this sample. The Spin Column can be reused but only for this sample.
2. Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube and discard the flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection Tube.
3. Wash the plasmid DNA
1. Add 750 μl of Column Wash Solution.
2. Centrifuge at maximum speed in a microcentrifuge for 1 minute at room
temperature.
3. Remove the Spin Column from the tube and discard the flowthrough.
4. Reinsert the Spin Column into the Collection Tube.
4. Wash again the plasmid DNA
1. Add 250 μl of Column Wash Solution.
2. Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room
temperature.
3. If the Spin Column has Column Wash Solution associated with it,
centrifuge again for 1 minute at maximum speed.
4. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being
careful not to transfer any of the Column Wash Solution with the Spin Column.
5. Elute the plasmid DNA
1. Add 100μl of Nuclease-Free Water to the Spin Column, wait 5 minutes
2. Centrifuge at maximum speed for 1 minute at room temperature in a
microcentrifuge.
6. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column.
7. Buffer
1. Add LTE Buffer (50ul/ 100ul)
8. Cap the microcentrifuge tube and store the purified plasmid DNA at –20°C or below.
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