Team:HIT-Harbin/notebook/protocol

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. 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.