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Team:Tsinghua/Protocol
From 2009.igem.org
This part will be revised and optimized gradually by Tsinghua iGEM09 team members.
Contents |
Microbiological Culture
General Protocol
Tips
Molecular Cloning
Isolation of Plasmid DNA
General Protocol
1)Bacteria culture: Inoculate the single colony harboring plasmid into 20 ml LB medium containing antibiotics, 37℃, 12-16 hours.
2)Pellet 1–3 ml of cells by centrifugation for 1–2 minutes at 12000rpm. Decant the supernatant. (1.4ml each time and collect about 3 ml culture of E.coli in 1.5ml eppendorf tube.) Completely resuspend the cell pellet in 100 μl solution 1 by vigorous vortex.
3)Add 150 μl solution 2, mix by inverting the tube 4-6 times gently and incubate the tube on ice for 1-2 minutes (do not exceed this period!). The cell suspension should be clear immediately.
4)Add 150 μl solution 3, invert the tube gently several times, place the tube at room temperature for 5minutes, and centrifuge at 12000rpm for 15 minutes.
5)Add 420 μl binding buffer to the mini-spin column. Then transfer the supernatant of procedure 3 to the same mini-spin column. Mix the supernatant and binding buffer with pipette carefully. Then place the column in another tube, centrifuge at 12000rpm for 30 and then discard waste liquid in the tube.
6)Add 750 μl wash buffer to the column, and centrifuge at 12000rpm for 1minute.
7)Repeat procedure 5. Then centrifuge at 12000rpm for 2minutes. Eliminate wash buffer as thoroughly as possible. The ethanol in wash buffer will impact the following enzyme-catalyzed reactions.
8)Carefully move the column into another clean tube. Add 50l Elution buffer or water into the column, place it at room temperature for 5minutes, andcentrifuge at 12000rpm for 1 minute.
Tips
1)Elution buffer should be added in the middle of adsorption material to guarantee all the plasmid DNA recovered.
2)To increase the recovery efficiency, increase the elution volume or elution times if it is necessary
3)0.5ml RNaseA（10mg/ml）can be added to the purified plasmid to eliminate RNA thoroughly.
4)If the molecular weigh of purified plasmid exceeds 10kb,the column may be placed in 70℃ water bath for 3-5 minutes to ensure plasmid DNA totally recovered.
Transformation of Recombinant DNA
General Protocol
1)Pipette competent cells suspension into the tubes, 100 μl each tube (If the competent cells are taken from -70℃, perform following steps immediately after thawing).
2)Add 10 μl of recombinant plasmid into the tube which is the tube with competent cells.
3)Mix the solutions gently, keep on ice for 20-30 minutes.
4)Heat shock by transferring the tubes to a water bath of 42℃ for 1 to 2 minutes.
5)Immediately return the tube to the ice bath. Keep on ice for 2 minutes.
6)Add 0.9 ml of LB (with no antibiotics added) into each tube. Incubate the tubes for 45 minutes to 1 hour at 37℃ to allow the cells to express their antibiotic gene product.
7)Spread about 200 μl of the resulting solutions (do dilution if necessary) on LB plates (with corresponding antibiotic added). After complete absorption of liquid LB, upside down the plates and incubate the plates at 37℃ overnight.
Tips
1)Never spread the transformation solution until you have assured that the glass stick is cooled down!
Ethanol Precipitation
General Protocol
1)Add 2 volume of 100% ethanol and 1/10 volume of 3M NaAc (pH=5.2) to the enzymatic digested solution.
2)Place the tube at -20℃ refrigerator for 20min.
3)Centrifuge at 4℃, 12,000r/min for 10 min.
4)Carefully discard the supernatant and add 100μl 70% ethanol.
5)Centrifuge and discard the supernatant again.
6)Desiccation at room temperature for 15 minutes.
7)Add 10 μl distilled water to dissolve DNA.
Tips
1)During centrifugation, the Ep tubes should be placed along a certain orientation, and you will save much time and energy when removing the supernatant.
Protein Isolation and Identification
Reference
Molecular Biology Section
[1] Sambrook J, Maniatis T, Fritsch EF. Molecular Cloning: a Laboratory Manual. cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd ed., 2001.
[2] Robert F. Weaver. Molecular Biology, McGrawHill, 4th edition, 2007.
