Chemically Competent E. coli Cells
autoclave 250mL LB in Erlenmeyer beaker
plate cells from cell stock on agarose plate with appropriate antibiotics
inoculate one clone in 5mL of LB with antibiotics, grow at 37°C o/n
expand to 500mL and grow until OD600nm = 0.35
transfer into 50mL Falcon tubes
refrigerate centrifuge at 4°C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol
spin down cells at 2000g for 10Min at 4°C
resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2
spin down at 2000g for 10Min at 4°C
resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol
freeze 50-100μl aliquots
store at 20°C
DoubleDigest Restriction of gBlocks and BioBricks
2μl 10x buffer (corresponding to enzymes)
0,5μl restriction enzyme 1
0,5μl restriction enzyme 2
1μg DNA
fill up to 20μl with H2O
Method:
mix all components
incubate at 37°C for 2h
heatinactivate enzymes at 80°C for 10min or use for gel purification
Gelpurification
This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega
Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube
Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060°C until gel slice is completely dissolved
Insert SV Minicolumn into Collection Tube
Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute
Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube
Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min
Discard flowthrough and reinsert Minicolumn into Collection tube
Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min
Empty the Collection Tube and centrifuge the column assembly for 1,5 min
Leave the tubes open for 10 min (to let any rest of ethanol evaporate)
Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube
Add 30μl of NucleaseFreeWater (65°C) to the Minicolumn. Incubate at room temperature for 10 min
Centrifuge at 16,000xg for 1 min.
Incubate sample at 65°C for 5min
Discard Minicolumn and store DNA at 4°C or 20°C
Smallscale plasmid preparation
Preparation was done using the PureYield™ Plasmid Miniprep System by Promega
Pick colonies for overnight cultures in 23ml LB medium with antibiotic
grow overnight
Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and discard supernatant
Resuspend the bacteria pellet in 600μl H2O
Add 100 μl Cell Lysis Buffer, invert six times
Add 350 μl cold Neutralization Solution, mix thoroughly by inverting
Centrifuge 30s at maximum speed
Transfer supernatant to PureYield Minicolumn in collection tube
Centrifuge for 30 sec, discard the flow-through
Add 200 μl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.
Add 400 μl Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.
Place column in eppendorf tube, add 30 μl Elution Buffer to minicolumn matrix, incubate for 1 min
Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA
Measure the DNA concentration using a Nanodrop
Transformation of Bacteria
50μl chemically competent E. coli
35μl overnight ligation mix OR 1ng purified plasmid DNA
Method:
Thaw bacteria pellet on ice
add ligation mixture or purified plasmid to bacteria
incubate for 20min on ice
heat shock bacteria for 45s at 42°C
incubate bacteria on ice for 2min
add 700μl LB medium without antibiotics
incubate for 1h at 37°C
spin down bacteria at 2g for 2min
discard supernatant by tipping the tube
resuspend bacterial pellet in leftover supernatant (50100μl)
streak bacteria onto LBagar plates with antibiotics and incubate overnight
