We are describing our research work. Below you can find the protocols we used.
plasmid | cassettes | ||
---|---|---|---|
psB1C3 | 1 | 2 | 3 |
psB1A3 | 1 | 2 | 3 |
psB1K3 | 1 | 2 | 3 |
2. 3A-assembly
Insert 1 E+S | Insert 2 X+P | Plasmid E+P |
---|---|---|
C1 | A1 | K1 |
A2 | K1 | C2 |
K3 | C2 | A3 |
C4 | A3 | K4 |
2. good to know before the start
3. Are you working with A. E.coli or B.yeast?
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 |
1. 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.
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 |
[1]https://www.highqu.com/media/wysiwyg/ressources/manuals/PCM02_ALLin_Red_Taq_Mastermix_PI.pdf
1. 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). |
2. Binding of DNA
3. Washing
4. Elution
– 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
2. Safty
3. What happens?
4. Assay conditions
5. Afterwards
1.Production of cleared lysate
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. completely resuspend the cell pellet by vortexing or pipetting
3. it is essential to thoroughly resuspend the cells
2. mix by inverting the tube 4 times - do not vortex
3. incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes)
2. mix by inverting the tube 4 times - do not vortex
3. incubate for 5 minutes at room temperature
2. immediately mix by inverting the tube 4 times - do not vortex
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.
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.
2. Centrifuge at maximum speed for 1 minute at room temperature in a microcentrifuge.
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