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Revision as of 11:08, 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
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- 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