Line 13: | Line 13: | ||
<div class="header_bottom_area_team"></div> | <div class="header_bottom_area_team"></div> | ||
<div class="main_content_area "> | <div class="main_content_area "> | ||
− | + | <div class=" content_area structure" > | |
− | + | <div class="single_header_title"> | |
− | + | <h1>Protocols</h1> | |
− | + | </div> | |
− | + | ||
− | + | <h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">General</h2> | |
− | + | <br/> | |
− | + | <ol id="category-tabs" style=" padding-left:0.1cm; list-style-type: none;"> | |
− | + | <li><a href="#" style="text-decoration: none; font-size: 18px"> | |
− | + | <b>Polymerase Chain Reaction (PCR)</b> <i class="fa fa-chevron-circle-down"></i></a> | |
− | + | <ol class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;"> | |
− | + | <li >Colony-PCR using Alkaline PEG</li> | |
− | + | <li> Colony-PCR with Heatshock</li> | |
− | + | <li>Extended Colony-PCR</li> | |
− | + | </ol> | |
− | + | </li> | |
+ | <li><a href="#" style="text-decoration: none; font-size: 18px"> | ||
<b>Precipitation</b> <i class="fa fa-chevron-circle-down"></i></a> | <b>Precipitation</b> <i class="fa fa-chevron-circle-down"></i></a> | ||
<ol class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;"> | <ol class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;"> | ||
Line 54: | Line 55: | ||
<li style=" padding-right: 0.3cm;"><span>conditions:</span></li> | <li style=" padding-right: 0.3cm;"><span>conditions:</span></li> | ||
<li style=" padding-right: 0.3cm;"><span>time: 3 min</span></li> | <li style=" padding-right: 0.3cm;"><span>time: 3 min</span></li> | ||
− | <li style=" padding-right: 0.3cm;"><span>pulse on: 30 s</span></li> | + | <li style=" padding-right: 0.3cm;"><span>pulse on: 30 s</span></li> |
− | <li style=" padding-right: 0.3cm;"><span>pulse off: 20 s</span></li> | + | <li style=" padding-right: 0.3cm;"><span>pulse off: 20 s</span></li> |
<li style=" padding-right: 0.3cm;"><span>amplitude: 50%</span></li> | <li style=" padding-right: 0.3cm;"><span>amplitude: 50%</span></li> | ||
Line 71: | Line 72: | ||
+ | <li><a href="#" style="text-decoration: none; font-size: 18px"> | ||
+ | <b>Transformation</b> <i class="fa fa-chevron-circle-down"></i></a> | ||
+ | <ol class="protocolhide" style=" padding-left:0.1cm; padding-right:0.1cm;list-style-type: none;"> | ||
+ | <li ><b>E. coli</b> | ||
+ | <ol type="1" style=" style=" padding-left:0.1cm; padding-right:0.1cm;align-content: center;"> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Thaw 100 µL <b>E. coli DH5α</b> or <b>E. coli BL21(DE3)</b> Gold on ice</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Add <b>DNA</b> (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; | ||
+ | 1-4 µL PLICing-reaction) to the competent cells and swirl gently</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Incubate for <b>15-30 min on ice.</b></span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Perform heatshock of the competent cells using a preheated water bath at <b>42°C for 45s</b> (DH5α, BL21).</li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Samples were immediately cooled down <b>5 min on ice.</b></span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Fill up the transformation mix <b>to 1 mL with SOC media</b> and | ||
+ | incubate at <b>37°C at 250rpm for 45 min </b>(45-60 min) for recovery of the cells.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span><b>Plate cells</b> on LB agar plate with antibiotic respectively and dry them under the clean bench. | ||
+ | <ol type="i" style=" padding-left: 0.5cm"> | ||
+ | <li><span>200 µL</span></li> | ||
+ | <li><span>resuspended pellet (from centrifugation of the leftover)</span></li> | ||
+ | </ol> | ||
+ | </span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Incubate the agar plates at <b>37°C overnight</b>(16-18 hours).</span></li> | ||
+ | <li><span>Count single colonies of each agar plate on the next day.</span></li> | ||
+ | </ol></li> | ||
+ | <li><b>Sonication</b> | ||
+ | <ol type="1"style=" style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" > | ||
+ | <li style=" padding-right: 0.3cm;"><span>Lorem ipsum dolor sit amet, consectetuer adipiscing elit.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Maecenas porttitor congue massa.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Fusce posuere, magna sed pulvinar ultricies, | ||
+ | purus lectus malesuada libero, sit amet commodo magna eros quis urna.</b></span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Nunc viverra imperdiet enim. Fusce est.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Nunc viverra imperdiet enim. Fusce est.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. | ||
+ | <ol type="i" style=" padding-left: 0.5cm"> | ||
+ | <li><span>Proin pharetra nonummy pede.</span></li> | ||
+ | <li><span>Mauris et orci.</span></li> | ||
+ | </ol> | ||
+ | </span></li> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | + | </ol></li> | |
− | + | ||
− | + | <li>Saccharomyces </li> | |
− | + | </ol> | |
− | + | </li> | |
− | + | <li><a href="#" style="text-decoration: none; font-size: 18px"> | |
− | + | <b>Preparation of Chemical Competent Cells </b> <i class="fa fa-chevron-circle-down"></i></a> | |
− | + | <ol class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;"> | |
− | + | <li ></li> | |
− | + | </ol> | |
− | + | </li> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | </ol> | ||
+ | <br/> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">Analytics</h2> | <h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">Analytics</h2> |
Revision as of 16:12, 7 October 2016
Protocols
General
-
Polymerase Chain Reaction (PCR)
- Colony-PCR using Alkaline PEG
- Colony-PCR with Heatshock
- Extended Colony-PCR
-
Precipitation
- test
-
Cell Lysis
- Lysozym
- Centrifuge the samples for 4 min at 4°C and 14000 rpm.
- Freeze the "dried" pellet overnight.
- Add 150 µL buffer on the pellet, resuspend it (e.g. with LB-medium) and incubate for 5 min at room temperature.
- Add 150 µL lysozym solution (c = 5-8 mg/mL).
- Incubate the samples for 1 h at 37°C and 250 rpm.
- Centrifuge the samples for 4 min at 4°C and 14000 rpm.
- Decant the supernatant.
- Sonication
- conditions:
- time: 3 min
- pulse on: 30 s
- pulse off: 20 s
- amplitude: 50%
- Glass beads
- Put some small glass beads into your sample tubes.
- Vortex your samples (5-10 min at level 2-6), avoid foam on the top of your sample.
- Put the used beads into a waste bottle filling with 70% EtOH for.
- Lysozym
-
Transformation
- E. coli
- Thaw 100 µL E. coli DH5α or E. coli BL21(DE3) Gold on ice
- Add DNA (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; 1-4 µL PLICing-reaction) to the competent cells and swirl gently
- Incubate for 15-30 min on ice.
- Perform heatshock of the competent cells using a preheated water bath at 42°C for 45s (DH5α, BL21).
- Samples were immediately cooled down 5 min on ice.
- Fill up the transformation mix to 1 mL with SOC media and incubate at 37°C at 250rpm for 45 min (45-60 min) for recovery of the cells.
- Plate cells on LB agar plate with antibiotic respectively and dry them under the clean bench.
- 200 µL
- resuspended pellet (from centrifugation of the leftover)
- Incubate the agar plates at 37°C overnight(16-18 hours).
- Count single colonies of each agar plate on the next day.
- Sonication
- Lorem ipsum dolor sit amet, consectetuer adipiscing elit.
- Maecenas porttitor congue massa.
- Fusce posuere, magna sed pulvinar ultricies, purus lectus malesuada libero, sit amet commodo magna eros quis urna.
- Nunc viverra imperdiet enim. Fusce est.
- Nunc viverra imperdiet enim. Fusce est.
- Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas.
- Proin pharetra nonummy pede.
- Mauris et orci.
- Saccharomyces
- E. coli
-
Preparation of Chemical Competent Cells
Analytics
-
Gelelectrophoresis
-
Analysis of Expression Level
- Preparation of pre-culture
- Fill 20 mL LB media into a (small) flask, add the corresponding antibiotics.
- Inoculate the pre-culture.
- Incubate your culture at 37°C, 250 rpm for 16-18 hours.
- Preparation of main culture
- Fill 200 mL LB media into a 1000 mL flask and add the corresponding antibiotics.
- Inoculate the main culture with 2-5 mL pre-culture, as to reach an OD600 of 0.1 in the main culture.
- Incubate your main culture at 37°C, 250 rpm till it reaches the OD600 of 1 (2-4 hours).
- Take a sample (500µL) out of your main culture and store it at -20°C.
- Induce the expression of your main culture by adding a final concentration of each 0.5 / 1 / 1.5 mM IPTG.
- Incubate at 37°C, 250 rpm for an hour.
- Take a sample hourly afterwards. (as described above)
- Analysis
- Perform a cell lysis with the samples which were taken and frozen at -20°C.
- Perform a Skim Milk Assay with every lysed sample.
- Perform a SDS-gel with all lysed samples.
- Preparation of pre-culture
-
SDS
-
Skim Milk Assay
-
AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay
Cloning
-
Enzymatic Digestion
-
Dephosphorylation
- Dephosphorylation after restriction
- Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) after restriction (example: 1 µL for 2000 kb) and according buffer (volume according to concentration of buffer).
- Incubate at 37°C for 30–60 minutes.
- Stop reaction by heat-inactivation of rSAP at 65°C for 5 minutes.
- Store at -20°C.
- Dephosphorylation after restriction
-
Ligation
-
Site Directed Mutagenesis (SDM) and Site Saturation Mutagenesis (SSM)
Media
Devices
Kits