Difference between revisions of "Team:Tsinghua/Protocol"

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Protocols

1. Plasmid DNA purification
(1)Centrifuge the 5mL culture tube for 10 minutes at 3000 rpm.
(2)Remove the culture medium and add 250uL buffer P1 (RNase added and 4°C stored) to resuspend the pelleted bacteria.
(3)Transfer to a 1.5mL EP tube.
(4)Add 250uL buffer P2 and gently mix by inverting the tube 8 times.
(5)Add 350uL buffer P3 and immediately mix by inverting the tube 8 times.
(6)Centrifuge the tube for 10 minutes at 12000 rpm.
(7)Transfer the supernatant to a pre-balanced (by buffer BL) column.
(8)Centrifuge the column for 60 seconds at 12000 rpm.
(9)Remove the flow-through and add 600uL buffer PW (alcohol added).
(10)Centrifuge the column for 60 seconds at 12000 rpm.
(11)Repeat step 9-10.
(12)Remove the flow-through and centrifuge for another time.
(13)Place the column in a 1.5mL EP tube and wait for 2 minutes at RT.
(14)Add 60uL ddH2O onto the column and centrifuge for 60 seconds at 12000 rpm.
(15)Measure the concentration of plasmid by Nanodrop.

2. Gel extraction
(1)Remove the gel containing DNA of interest and place it into a 1.5mL EP tube.
(2)Add 500uL solution SN per 100mg agarose gel and dissolve the gel for 5 minutes at 65°C.
(3)Add 50uL solution B per 500uL solution SN and mix them up.
(4)Place the 3S column into the collecting tube and transfer the solution mixture into the column.
(5)Place the column for 2 minutes at RT and then centrifuge for 1 minute at 10000 rpm.
(6)Discard the flow-through and add 600uL Wash solution.
(7)Centrifuge for 1 minute at 10000 rpm.
(8)Repeat step 6-7.
(9)Place the column into a new 1.5mL EP tube and add 30uL ddH2O.
(10)Place the column for 2 minutes at RT and centrifuge for 1 minute at 10000 rpm.
(11)Measure the concentration of DNA by Nanodrop.

3. Chemical transformation
(1)Add 5uL plasmid into 50uL competent DH5α and place the tube on ice for 30 minutes.
(2)Heat up the tube for 90 seconds at 42°C.
(3)Place the tube on ice for 2.5 minutes.
(4)Add 1mL LB (without antibiotics) on a germ-free bench.
(5)Recover the bacteria by shaking the tube for 30 minutes at 37°C.
(6)Centrifuge (if needed) for 3 minutes at 5000 rpm and remove most of the supernatant.
(7)Resuspend the bacteria and add 100uL suspension onto the plate with antibiotics.
(8)Culture overnight at 37°C.

4. Transformation protocol for distribution kit
(1)Insert a tip into the well containing plasmid of interest.
(2)Add 10uL ddH2O and dissolve the plasmid for 5 minutes.
(3) 5uL for transformation.

5. Electro-transformation
(1)Produce electroporation-competent cells.
(2)Add 50uL bacteria into 100mL LB and culture at 30°C until its optical density (OD600) reaches 0.05-0.1. Then add 1mM L-arabinose until it reaches 0.6. Place the cells on ice for 1 hour and then wash with overnight-stored cold H2O twice and cold 10% glycerol once. Condense 100 times and dispense 50uL into each tube. Store at -80°C.
(3)Electro-transform 10-100ng DNA per 50uL cells using Eporator from Eppendorf with 1mm3 electric shock cup (Electric shock is given at 1.8kV for 4-5 seconds).
(4)Recover the bacteria in 1mL LB at 37°C.
(5)Plate the bacteria on the plate with antibiotics.

6. PCR
(1)Commonly used system:

ddH2O add up to 10uL
Total volume: 10uL

(4)Place the reaction mix for 15 minutes at 50°C.
(5)Use 5uL mix for transformation.
Note: Ratio between inserts and ratio between vector and inserts should be kept at 1:1 and 1:3 respectively.

12. Bacterial genome one-step knock-out assay
Adapted from method designed by Wanner.
Prepare the following plasmids:

	Requirement from the host bacteria	Resistance gene	Temperature requirement
pKD46		Amp	30°C
pKD3/4/13	BW25141	Amp, Kan or Cm
pCP20		Amp, Cm	30°C

1. Transform pKD46 (temperature-sensitive, culture at 30°C) and screen on Amp+ plate.
2. Extract the plasmid and confirm.
3. Amplify the Kanamycin gene to be inserted with primers share homology (usually 39bp for homologous arm and 20bp for matching sequence of Kan) to the genome by PCR.
4. Electro-transform the fragment after PCR and screen on Kan+ plate.
5. Pick multiple colonies and preserve on Kan+ plate.
6. Meanwhile confirm the colonies by colony PCR.
7. Culture in LB at 37°C without antibiotics overnight in order to remove pKD46.
8. Culture on plate at 30°C without antibiotics.
9. Pick colony and screen positively using Kan+ plate.

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