- 西亚试剂：：Preparing Overnight Bacterial Culture
Sterile LB medium (Luria-Bertani Medium) with or without antibiotic:
water 500 ml
bacto-tryptone 10 g
bacto-yeast extracts 5 g
sodium chloride 10 g
stir to dissolve
pH to 7.0
autoclave to sterilize
cool to 50-60 degrees C
add antibiotic if needed
Agar plate with bacterial colonies:
scrape bacteria glycerol stock such as E coli DH5 (LifeTechnologies) or scratch a single colonies from an old agar plate with bacteria...详细+
- 西亚试剂：：Plasmid Quickpreps
grow up single colony in 1.5 ml LB/antibiotic ovn @37°C
pour into tube, spin for 30 sec
decant supernatant and resuspend pellet in 100 ml lysis solution
add 200 ml alkaline SDS, vortex well
incubate for 5 min on ice
add 150 ml high salt solution, vortex well
incubate for ca. 10 min on ice
spin for 10 min
remove 400 ml supernatant and transfer to new tube prefilled with 400 ml iso...详细+
- 西亚试剂：：Quick DNA Plasmid Prep
Quick DNA Plasmid Prep
This protocol gives very clean plasmid preps for restriction digests and cloning. However, due to the alkaline lysis step, the DNA is often nicked and may not give exceptional sequence data.
0.1 N NaOH 1 ml 10N NaOH
0.2% SDS 1 ml 20% SDS
10 mM Tris 7.5 1 ml 1M Tris 7.5
1 mM EDTA 200 ul 0.5 M EDTA
up to 100 ml with Q
store at room temperature
3 M NaOAc 5.2
24.6 g anhydrous...详细+
- 西亚试剂：：CsCl Prep of Plasmid DNA
CsCl Prep of Plasmid DNA
This is a standard large scale prep. for plasmid DNA which gives a yield of 0.5 - 1.0 mg. I have made some minor changes to the MHB protocol.
Solution I, II, III from protocol D.1.
Tris/EDTA pH 7.5 (optional)
20 ml 1 M Tris 7.5
4 ml 0.5 M EDTA 8.0
up to 2 liters with Q
store at 4 degrees for dialysis
Dialysis Tubing (optional)
The tubing is stored dry at 4 d...详细+
- 西亚试剂：：DNA Plasmid Maxiprep Protocol
DNA Plasmid Maxiprep Protocol
1. Pick single colony and inoculate 250 ml ofcontaining 100 mg/l ampicillin or appropriate antibiotic. Shake at 250 RPM overnight.
2. Centrifuge cells in a Sovall GSA (250 ml)or SLA-3000 (500 ml) rotor at 5 k × g for 10 minutes.
3. Resuspend cell pellet in 5 ml of (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8) by pipetting up and down with a 10ml pipette. Optional: add a spatula tip of lysozyme powder. A...详细+
Plasmid stability test
Immediately before indcution, it is advisable to test the culture to determine the fraction of cells that still carry the target plasmid. This involves plating of cells on four different plates.
cells that grow on these plat...详细+
- 西亚试剂：：Screening of recombinants
Screening of recombinants
It is best to do a test ligation without insert, if the background is low, it would not be necessary to screen colonies for insert.
1) colony PCR
1) Pick a colony from plate and suspend the colony in 10 µl of 1X PCR buffer. Spot a fresh plate (divided into squares and numbered) with a drop of this suspension (or inoculate plate first then resuspend the rest in buffer). You may do as many as you wish (typically 8 -...详细+
- 西亚试剂：：Stable Transfection (Electroporation)
Stable Transfection (Electroporation)
Electroporation can be used for both transient and stable transfection of mammalian cells. Cells are placed in suspension in an appropriate electroporation buffer and put into an electroporation cuvette. DNA is added, the cuvette is connected to a power supply, and the cells are subjected to a high-voltage electrical pulse of defined magnitude and length. The cells are then allowed to recover b...详细+
- 西亚试剂：：Electroporation of P. aeruginosa
Electroporation of P. aeruginosa
Preparation of Electrocompetent Cells
Inoculate a 5-ml of L-broth with cells of the P. aeruginosa strain to be electrotransformed. Grow culture overnight at 37°C on a roller.
Transfer 2 ml of the overnight culture to 200 ml of fresh L-broth in a 1-liter sidearm flask. Grow this culture at 37°C with shaking (200 rpm) until...详细+
- 西亚试剂：：How to Make Competent Cells
How to Make Competent Cells
Step 1: Take an 100 mL aliquot of frozen cells from the -80oC and innoculate about 500 ml to 1 L sterile LB broth. DO NOT ADD antibiotic, since these cells do not have an plasmid in them. Work as sterile as possible.
Step 2: Grow the cells on a shaker at 37oC until they reach an OD @ 600nm of 0.3 to 0.4 (1 cm pathlength).
Step 3: Centrifuge in the Sorvall GSA rotor (250 ml centrifuge bottle) at 5,000 RPM for 10 minu...详细+