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Team:HIT-Harbin/notebook/protocol
From 2012.igem.org
Yeast extact 5g
Peptone 10g
NaCl 10g
Distilled water 1000ml pH 7.0
Range of application: Escherichia.coli
Yeast extact 5g
Peptone 10g
NaCl 10g
Agar 1-2%
Distilled water 1000ml pH 7.0
Boil the mixture in autoclave at 121℃ for 30min
Range of application: Escherichia.coli
1. Place one colony in 3~5mL LB media, grow overnight at 37℃;
2. Transfer overnight DH5α culture(1:100~1:50)into 100mL LB liquid media;
3. Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (2-3 hours);
4. Place cells on ice for 10 min;
5. Centrifuge cells at 4℃ for 10 min at 3,000g;
Subsequent resuspensions is done in the same bottle. Cells remain cold for the rest of the procedure: transport tubes on ice and resuspend on ice in the cold room.
6. Pour off media and resuspend cells in 10mL cold 0.1 M CaCl2, and incubate on ice for 30 min;
7. Centrifuge at 4℃ for 10 min at 3,000g;
8. Pour supernatant and resuspend cells (by pipetting) in 4mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 100 μL into 1.5mL centrifuge tubes placed on ice. Cells is stored at -80℃ and can be used for transformation for up to 6 months.
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Prepare the following reaction mixture:
2. Incubate 10 min at 22℃
3. Use up to 5μL of the mixture for transformation of 50μL of chemically competent cells.
10μL 5x buffer(Mg2+ plus)
4μL dNTPs
1.0μL forward primer
1.0μL reverse primer
1.0μL template (10pg-1ng)
1.0μL DNA polymerase
ddH2O up to 50.0μL
Note: Based on primers, set an appropriate annealing tem.
Keep everything on ice and add all volumes in a PCR tube.
1. Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA;
2. Place the gel in the apparatus rig with the wells facing the negative end (black-colored);
3. Fill the rig with 1x TBE buffer;
4. Load 2μL of 1kb ladder;
5. Add 2μL of 6x loading dye to each PCR reaction tube, adn load 20μL in each well;
6. Run at 120V.
1. Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice;
2. Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel(100mg = 100μL);
3. Dissolve the gel slice using a 60μC heat block;
4. Apply the dissolved gel to the QIAquick column and centrifuge at 13,000rpm for 1 minute;
5. Discard the flow-through and repeat Step 4 until all sample has passed through the column;
6. Add 750μL of rinse buffer to the QIAquick column and centrifuge at 13,000rpm for 1 minute;
7. Add 500L of rinse buffer to the QIAquick column and centrifuge at 13,000rpm for 1 minute;
8. Wash the column with 750μL of Buffer PE and centrifuge at 13,000rpm for 1 minute;
9. Discard the flow-through and centrifuge at 13,000rpm for 2 minute to remove residual EtOH;
10. Transfer the QIAquick column to a new Eppendorf;
11. Add 50μL elution buffer to the center of the column and wait at least 2 minutes;
12. Centrifuge at 13,000rpm for 1 minute.
