Paulinchen (Talk | contribs) |
Paulinchen (Talk | contribs) (protocols) |
||
Line 503: | Line 503: | ||
<div class="spoiler"> | <div class="spoiler"> | ||
<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Salkowski Assey" /> | <input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Salkowski Assey" /> | ||
− | <div class="inner" style="display:none;"> | + | <div class="inner" style="display:none;"> |
+ | |||
+ | Salkowski Assay for colony screening | ||
+ | Large qualitative screening of IAA-producing colonies at the same time to see if our constructs are still functional in our E.coli/yeasts | ||
+ | Helps us pic kthe right colonies for colony-PCR and GC-MS measurements | ||
+ | Reagent: 2% 0.5M FeCl3 in 35% perchloric acid | ||
+ | Perchloric acid is highly corrosive and dangerous!! Always uses protective gear and work under a fume hood! | ||
+ | Reagent will always be mixed together on the spot, FeCl3 stock solution is finished, acid will be taken from the chemicals sheld from the AG plant physiology (has been negotiated) | ||
+ | What happens? | ||
+ | Reagent reacts to IAA (and other indolic compounds) to make several colored products | ||
+ | IAA will be seen as bright red (other compounds brown or yellowish) | ||
+ | Assay conditions | ||
+ | Plates were inoculated in a grid pattern and overlaid with an 82mm-diameter disk of Nitrocellulose membranes | ||
+ | Plates are overlaid with Nitrocellulose immediately after inoculation with toothpicks | ||
+ | After normal incubation (i.e. overnight) time, the membrane was removed and soaked in reagent (or reagent-saturated [2.5 mL] filter paper, here “Whatman grade 2” had best results)) | ||
+ | After 30 - 60 minutes, coloring reaction is finished and fading began | ||
+ | Best results with colony sizes between 0.5 to 2 mm | ||
+ | Addition of Tryptophan greatly enhances color reaction but does not interfere with distinguishing IAA positive and negative colonies (yellow background and strong red to pink positives) | ||
+ | Other indolic compounds (i.e. indolepyruvic acid) are distinguishable by a more yellow-brownish color | ||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
Line 511: | Line 533: | ||
<div class="spoiler"> | <div class="spoiler"> | ||
<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (E.Coli)" /> | <input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (E.Coli)" /> | ||
− | <div class="inner" style="display:none;"> Protocol is | + | <div class="inner" style="display:none;"> |
+ | |||
+ | Tranformation Protocol | ||
+ | What is it? | ||
+ | Transmission of genetic information into competent cells (or plants, algas, mushrooms) (the target organismn) | ||
+ | Übertragung von genetischer Information durch Aufnahme freier DNA in kompetente Zellen oder auch in Pilzen/Algen/Pflanzen | ||
+ | DNA in Zielorganismen einzuschleusen | ||
+ | Why we are doing it? | ||
+ | Transmission of genetic information into competent cells | ||
+ | What you need: | ||
+ | *1.5 mL tube, pipette (50µL), pipette(1000µL), plate | ||
+ | *Ice, SOC Medium, competent cells | ||
+ | How we are doing it? | ||
+ | 1. Prechil 1.5 ml tube on ice | ||
+ | |||
+ | 2. Thaw a tube competent E. coli cells on ice for 10 minutes. | ||
+ | |||
+ | 2.1. mix gently | ||
+ | 2.2. Pipette 50 µl of the cells into the 1.5ml tube | ||
+ | |||
+ | (Temperature over 0°C decrease the efficiency of the transformation!) | ||
+ | |||
+ | 3. Add 1-5 µl (containing 1 pg-100 ng of plasmid) DNA to the cell mixture. | ||
+ | |||
+ | (as soon as as the last bit of ice in the tube is disappeared!) | ||
+ | |||
+ | 4. Flick the tube 4-5 times to mix cells and DNA. | ||
+ | |||
+ | (No vortexing!) | ||
+ | |||
+ | 5. Place the mixture on ice for 30 minutes | ||
+ | |||
+ | (without mixing!) | ||
+ | (2-fold loss in transformation efficiency for every 10 minutes this step is shortened!) | ||
+ | |||
+ | 6. Heat shock at exactly 42°C for exactly 30 seconds. | ||
+ | |||
+ | (without mixing!) | ||
+ | (temperature and timing specific to transformation volume and vessel) | ||
+ | |||
+ | 7. Place on ice for 5 minutes. | ||
+ | |||
+ | (without mixing!) | ||
+ | |||
+ | 8. Pipette 950 µl of room temperature SOC into the mixture. | ||
+ | |||
+ | 9. Place at 37°C for 60 minutes and shake vigorously (800 rpm in thermo mix block) | ||
+ | |||
+ | (2-fold loss in transformation efficiency for every 15 minutes this step is shortened) | ||
+ | (SOC gives 2-fold higher transformation efficiency than LB medium) | ||
+ | (incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than without) | ||
+ | |||
+ | |||
+ | 10. Warm selection plates to 37°C | ||
+ | |||
+ | (plates can be used warm or cold, wet or dry…efficiency is nearly the same… warm plates easier to spread and allow most rapid colony formation) | ||
+ | |||
+ | 11. Mix the cells thoroughly by flicking the tube and inverting | ||
+ | |||
+ | 12. Spread 50-200 µl onto a selection plate and incubate overnight at 37°C. | ||
+ | |||
+ | (Alternative: incubate at 30°C for 24-36 hours or 25°C for 48 hours.) | ||
+ | (For low efficiency cloning reactions: spin down the whole transformation mixture, remove nearly the complete supernatant (approx. 900 ul), resuspend cells in remaining liquid and plate completely) | ||
+ | |||
+ | </div></div> | ||
Line 519: | Line 605: | ||
<div class="spoiler"> | <div class="spoiler"> | ||
<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (yeast)" /> | <input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (yeast)" /> | ||
− | <div class="inner" style="display:none;"> | + | <div class="inner" style="display:none;"> |
+ | |||
+ | Zymo Research: | ||
+ | Preparation of Competent Cells | ||
+ | Grow yeast cells at 30 | ||
+ | ° | ||
+ | C in 10 ml YPD broth until mid-log phase (~5 x 10 | ||
+ | 6 | ||
+ | - 2 x 10 | ||
+ | 7 | ||
+ | cells/ml or OD | ||
+ | 600 | ||
+ | of 0.8-1.0). The | ||
+ | following steps are accomplished at room temperatur | ||
+ | e. | ||
+ | 1. Pellet the cells at 500 x g for 4 minutes and di | ||
+ | scard the supernatant. | ||
+ | 2. Add 10 ml | ||
+ | EZ 1 solution | ||
+ | to wash the pellet. Repellet the cells and discard | ||
+ | the supernatant. | ||
+ | 3. 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 bel | ||
+ | ow -70 | ||
+ | ° | ||
+ | C for future | ||
+ | use. It is important to freeze the cells slowly. | ||
+ | To accomplish this, either wrap the aliquotted cell | ||
+ | s in 2-6 layers of paper | ||
+ | towels or place in a Styrofoam box before placing i | ||
+ | n the freezer. | ||
+ | DO NOT | ||
+ | use liquid nitrogen to snap-freeze the cells. | ||
+ | Transformation | ||
+ | This part of the procedure is the same for both fro | ||
+ | zen stored (thawed at room temperature) and freshly | ||
+ | prepared | ||
+ | competent yeast cells. | ||
+ | 1. Mix 50 | ||
+ | μ | ||
+ | l of competent cells with 0.2-1 | ||
+ | μ | ||
+ | g DNA (in less than 5 | ||
+ | μ | ||
+ | l volume); add 500 | ||
+ | μ | ||
+ | l | ||
+ | EZ 3 solution | ||
+ | and mix thoroughly. | ||
+ | 2. Incubate at 30 | ||
+ | ° | ||
+ | C for 45 minutes. Mix vigorously by flicking with f | ||
+ | inger or vortexing (if appropriate for | ||
+ | your DNA) 2-3 times during this incubation. | ||
+ | 3. Spread 50-150 | ||
+ | μ | ||
+ | l of the above transformation mixture on an appropr | ||
+ | iate 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 | ||
+ | . | ||
+ | |||
+ | </div></div> | ||
Line 527: | Line 680: | ||
<div class="spoiler"> | <div class="spoiler"> | ||
<input type="button"style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Ligation" /> | <input type="button"style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Ligation" /> | ||
− | <div class="inner" style="display:none;"> Protocol | + | <div class="inner" style="display:none;"> |
+ | |||
+ | Ligation Protocol with T4 DNA Ligase (M0202) | ||
+ | 1. | ||
+ | Set up the following reaction in a microcentrifuge tube on ice. | ||
+ | T4 DNA Ligase Bu | ||
+ | ff | ||
+ | er (10 x) | ||
+ | 2 ul | ||
+ | T4 DNA Ligase | ||
+ | 1 ul | ||
+ | Vector DNA | ||
+ | Insert DNA | ||
+ | Nuclease-free water | ||
+ | to 20 ul | ||
+ | 1. | ||
+ | Calculation of the DNA | ||
+ | 2. | ||
+ | Example calculation | ||
+ | 1:3 vector to insert | ||
+ | mass Vector DNA: 100 ng | ||
+ | Vector DNA: 10 kb | ||
+ | Insert DNA: 3 kb | ||
+ | 2. | ||
+ | T4 DNA Ligase should be added last. | ||
+ | 3. | ||
+ | Use | ||
+ | nebiocalculator.neb.com/#!/ | ||
+ | to calculate molar ratios. | ||
+ | 4. | ||
+ | The T4 DNA Ligase Bu | ||
+ | ff | ||
+ | er should be thawed and resuspended at room temperature. | ||
+ | 2. | ||
+ | Gently mix the reaction by pipetting up and down and microfuge briefly. | ||
+ | 3. | ||
+ | Incubation | ||
+ | 1. | ||
+ | cohesive (sticky) ends | ||
+ | 1. | ||
+ | 16°C overnight or room temperature for 10 minutes. | ||
+ | 2. | ||
+ | blunt ends or single base overhangs | ||
+ | 1. | ||
+ | 16°C overnight or room temperature for 2 hours | ||
+ | (alternatively, high concentration T4 | ||
+ | DNA Ligase can be used in a 10 minute ligation) | ||
+ | . | ||
+ | 4. | ||
+ | Heat inactivate at 65°C for 10 minutes. | ||
+ | 5. | ||
+ | Chill on ice and transform 1-5 | ||
+ | μ | ||
+ | l of the reaction into 50 | ||
+ | μ | ||
+ | l competent cells | ||
+ | |||
+ | </div></div> | ||
Line 535: | Line 745: | ||
<div class="spoiler"> | <div class="spoiler"> | ||
<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Miniprep"/> | <input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Miniprep"/> | ||
− | <div class="inner" style="display:none;"> | + | <div class="inner" style="display:none;"> |
+ | |||
+ | Promega “Wizard Plus SV Miniprep Purification System“ | ||
+ | mehr Infos zum Kit sind in der Anleitung zu finden (ausgedruckt hinter dem “Miniprep-Protokoll“ | ||
+ | 1. | ||
+ | Production of cleared lysate | ||
+ | 1. | ||
+ | Isolation of the bacteria | ||
+ | 1. | ||
+ | harvest 1–5ml (high-copy-number plasmid) or 10ml (low-copy-number plasmid) | ||
+ | of bacterial culture | ||
+ | 2. | ||
+ | centrifugation for 5 minutes at 10,000 x | ||
+ | g | ||
+ | in a tabletop centrifuge | ||
+ | 3. | ||
+ | pour o | ||
+ | ff | ||
+ | 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 | ||
+ | μ | ||
+ | l 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 | ||
+ | μ | ||
+ | l 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 | ||
+ | μ | ||
+ | l 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 | ||
+ | μ | ||
+ | l 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 | ||
+ | |||
+ | </div></div> | ||
Revision as of 20:09, 25 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
Main sponsors: