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<b>Cell Lysis</b> <i class="fa fa-chevron-circle-down"></i></a> | <b>Cell Lysis</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;"> | ||
− | <li >Lysozym</li> | + | <li><b>Lysozym<b> |
+ | <ol type="1" style=" padding-left:0.1cm; padding-right:0.1cm;align-content: center;"> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Centrifuge the samples for <b>4 min</b> at <b>4°C</b> and <b>14000 rpm</b>.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Freeze the "dried" pellet overnight.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Add <b>150 µL</b> buffer on the pellet, resuspend it (e.g. with LB-medium) and incubate for <b>5 min</b> at room temperature.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Add <b>150 µL</b> lysozym solution <b>(c = 5-8 mg/mL)</b>.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Incubate the samples for <b>1 h</b> at <b>37°C</b> and <b>250 rpm</b>.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Centrifuge the samples for <b>4 min</b> at <b>4°C</b> and <b>14000 rpm</b>.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Decant the supernatant.</span></li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </li> | ||
<li> Sonication</li> | <li> Sonication</li> | ||
<li>Glass beads</li> | <li>Glass beads</li> |
Revision as of 20:08, 6 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
- Glass beads
- 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
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- Saccharomyces
- E. coli
-
Preparation of Chemical Competent Cells
Analytics
-
Gelelectrophoresis
-
Analysis of Expression Level
-
SDS
-
Skim Milk Assay
-
AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay
Cloning
-
Enzymatic Digestion
-
Dephosphorylation
-
Ligation
-
Site Directed Mutagenesis (SDM) and Site Saturation Mutagenesis (SSM)
Media
Devices
Kits