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 |
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
DNA Purification with the Wizard® SV Gel and PCR Clean-Up System
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). |
3. Washing
4. Elution
[1]https://www.promega.de/-/media/files/resources/protcards/wizard-sv-gel-and-pcr-clean-up-system-quick-protocol.pdf?la=de-de
2. Why are we doing it ?
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.
1. Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins.
2. 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.
Note: Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. If you add EtBr to your gel, you will also want to add it to the running buffer when you run the gel.
3. Pour the agarose into a gel tray with the well comb in place.
Note: Think about witch gel tray size you need. (a small one or a big one.)
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.
4. Let the newly poured gel sit at room temperature for 20-30 mins, until it has completely solidified.
5. Loading Samples and Running an Agarose Gel:
1. 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: 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.
6.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.
7. 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.Preparation of Competent Cells
(~5 x 106 - 2 x 107 cells/ml or OD 600 of 0.8-1.0).
The following steps are accomplished at room temperature.
3. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
4. Add 1 ml EZ 2 solution to resuspend the pellet. At this point, the competent cells can be used for transformations directly or stored frozen at or below -70°C for future use. It is important to freeze the cells slowly. To accomplish this, either wrap the aliquotted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer.DO NOT use liquid nitrogen to snap-freeze the cells.
2. Transformation
2. Incubate at 30 °C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
3. Spread 50-150 μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants.
[1] http://www.zymoresearch.com/downloads/dl/file/id/165/t2001i.pdf
1. Set up the following reaction in a microcentrifuge tube on ice.
3.Incubation
5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells
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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