Line 59: | Line 59: | ||
</ol></li> | </ol></li> | ||
− | <li>Glass beads</li> | + | <li><b>Glass beads</b> |
+ | <ol type="1" style=" padding-left:0.1cm; padding-right:0.1cm;align-content: center; "> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Put some <b>small</b> glass beads into your sample tubes.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Vortex your samples <b>(5-10 min at level 2-6)</b>, <b>avoid foam</b> on the top of your sample.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Put the used beads into a waste bottle filling with <b>70%</b> EtOH for.</span></li> | ||
+ | </ol> | ||
+ | </li> | ||
</ol> | </ol> | ||
</li> | </li> |
Revision as of 20:21, 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
- 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
-
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