Difference between revisions of "Team:USTC/Notebook"

1.Set up the following reaction on ice:

2.Vortex briefly.

3.Incubate at 37°C for at least 60 minutes.

To make a 1% agarose gel:
1.Weigh out 0.4g of agarose powder
2.Transfer to a microwave bottle
3.Pour 40mL of 1x TAE buffer into the bottle
4.Microwave until clear
5.Pour the gel and let it polymerize until it is solid.

1.Add the appropriate 6x DNA loading Buffer to the PCR or digestion reaction mixture.
2.Place the gel into the electrophoresis tank and pour 1x TAE buffer
3.Pipet the DNA into the gel pockets.
4.Run the gel at 120V for 30 min.
5.Image the gel using ultraviolet light. If necessary, cut out bands.

1.Perform agarose gel electrophoresis to fractionate DNA fragments.
2.Minimize the size of the gel slice by removing extra agarose.
3.Add 1 volume Binding Buffer (XP2).
4.Incubate at 50°C-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
5.Insert a HiBind DNA Mini Column in a 2 mL Collection Tube.
6.Add no more than 700 μL DNA/agarose solution from Step 4 to the HiBind DNA Mini Column.
7.Centrifuge at 10,000g for 1 minute at room temperature.
8.Discard the filtrate and reuse collection tube.
9. Repeat Steps 6-8 until all of the sample has been transferred to the column. Add 300 μL Binding Buffer (XP2).
10. Centrifuge at maximum speed for 1 minute.
11. Discard the filtrate and reuse collection tube.
12.Add 700 μL SPW Wash Buffer.Centrifuge at maximum speed for 1 minute at room temperature.Discard the filtrate and reuse collection tube.
13.Repeat Step 12 once.
14.Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
15.Lying for 10 min.
16.Put the HiBind DNA Mini Column in a new EP tube. Add 30 μL 50°C ddH2O, 10 min's standing, maximum speed centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

1.Take the competent bacteria from refrigerator and incubate them into ice about 5 mins until it is dissolved
2.Absorb 100pg to 10 ng plasmid and mix it with bacteria solution thoroughly. ATTENTION: Please operate this step tenderly!!!
3.Put the tubes on the ice about 30 mins.(Time SHOULD BE ACCURATE)
4.Make a heat shock at 42 degree centigrade about 90 sec (TIME SHOULD BE ACCURATE)
5.Put the tubes on the ice about 5 mins again.
6.Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 1 hours .
7.Centrifuge them at 4000 rpm about 2 mins and we will see sediment in the tubes.
8.Discard the supernatant liquid and leave about 200 ul medium.
9.Coat plate: add 200 ul solution in a large plate.
10.Cultivate these bacteria overnight for further use.

1.Pick up some bacterial solution with the inoculating loop and inoculate it onto the anti-LB plate by plate scribing.
2.Incubate at 37⁰C overnight.
3.Pick colonies in 5 mL EP tubes without anti-LB liquid mediumIncubate at 37⁰C, shaking at 250rpm for 6 hours.
4.Transfer 2 mL of bacterial solution to 200 mL of LB liquid medium at 37 ° C shaker until the OD value reaches 0.4.
5.Transfer the medium to a 50 mL pre-cooled centrifuge tube and place on ice for 30 min.
6.Spin down for 15 minutes at 4000rpm at 4⁰C.
7.Discard supernatant, resuspend pellet in 30ml of 80mM CaCl2-20mM MgCl2, keep on ice.
8.Spin down for 15 minutes at 2700rpm at 4⁰C.
9.Discard supernatant, resuspend pellet in 2mL 100mM CaCl2-18% glycerin, keep on ice，then dispense to EP tube.
10.Store at -80℃ for further use.

1.Set up the following reaction on ice:

2.Vortex thoroughly and spin briefly.

3.Incubate the mixture at 16℃ overnight.

1.Set up the following reaction on ice:

2.PCR Conditions

3.Store at 4℃ for further use.

1.Transfer the PCR reaction or enzymatic reaction to a clean 1.5 ml centrifuge tube and add 5 volumes Buffer B3, mix well.
2.Transfer all the mixture to the adsorption column and centrifuged at 8,000 × g for 30 sec, discard filtrate.
3.Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once.
4.12000 rpm centifuge 1 min.
5.Lying for 10 min.
6.Put the adsorption column in a new EP tube. Add 20~30 μL 50°C ddH2O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

*Optimized cloning efficiency is 50 ng of vectors with 3 fold of excess inserts.

2.Incubate at 50°C for 30 minutes to 1 hour, depending on number of fragments being assembled.

3.Transform 4 µl of the reaction mixture into competent E. coli, or use the mixture directly in other applications.

1.Add bacteria in 500mL LB medium, shake in 37℃ to OD600 reach 0.8
2.Add 500μL 1M IPTG, cultivate in (temperature) for (time)
3.Centrifuge at 8000 rpm, 4℃ for 10 min, then remove the supernatant
4.Repeat step 3 untill all the bacteria have been collected
5.Resuspend the bacteria with 50 mL TBS
6.Centrifuge at 8000 rpm, 4°C for 10 min, then remove the supernatant
7.Resuspend the bacteria with 15 mL lysis buffer
8.The cells were disrupted via ultrasonication (Power 35%, 30min, total duty in cycles of 1s on, 2s off)
9.Centrifuge at 14000 rpm, 4°C for 20 min
10.Retrieve the supernatant for future experiments.

1.Pure out 20% ethanol inside of the tube
2.Wash the tube with 10mL 1M imidazole 3 times
3.Wash the tube with 10mL H2O
4.Add 10mL 100mM NiCl into the tube, then release the solution after a few minutes
5.Wash the tube with 10mL H2O
6.Balance the tube with 10mL lysis buffer 3 times
7.Add the cell lysis in the tube (flow rate: 1~1.5mL/min)
8.Wash the tube with 10mL lysis buffer
9.Wash the tube with 10mL buffer contained 20、50、80mM imidazole
10.Wash the tube with 10mL elution buffer and collect
11.Wash the tube with 10mL 1M imidazole 3 times
12.Wash the tube with 10mL H2O
13.Add 5mL 20% ethanol to storage