Team:HIT-Harbin/Protocol

1.The composition of LB medium for E. coli

1)Liquid medium (1L)

NaCl 10g

Tryptone 10g

Yeast extract 5g

Distilled water 1000ml

2)Solid medium

NaCl 10g

Tryptone 10g

Yeast extract 5g

Distilled water 1000ml

Agar powder 10~20g

Sterilize the mixture in autoclave at 121℃ for 30 minutes.

2.The preparation of competent E.coli cell (DH5α)

1)pick one colony and put it in 3~5ml LB media , overnight at 37℃

2)Transfer the centrifugal sediment into 100ml LB liquid media

3)Grow the cuLture at 37℃(250rpm), until OD600 = 0.4 (2-3h)

4)Place cuLture on ice for 10 minutes

5)Centrifuge cuLture at 4℃ for 10 minutes at 4,000 rpm Subsequent resuspensions is done in the same tube. CuLture remains cold for the rest of procedure. Place tubes on ice and resuspend the cuLture the on ice

6)Remove the media and resuspend cells in 10ml cold 0.1 M CaCl2 and incubate it on ice for 30 minutes

7)Centrifuge it at 4℃ for 10 minutes at 3,500 rpm

8)Remove the supernatant and resuspend cells (by pipetting) in 4 ml cold 0.1 M CaCl2 containing 15% glycerol. Transfer 100uL the mixture into 1.5 ml centrifuge tubes placed on ice. Cells are stored at -80℃ and can be used for transformation in 6 months

4.Restriction enzyme reaction system

1)formula

2)Mix gently and centrifuge it for a few seconds

3)Incubate it at 37℃ for 5 minutes

The combination of double enzyme digestion are as following:

FD EcoRⅠ、FD Pst Ⅰ

FD XbaⅠ、FD Pst Ⅰ

FD EcoRⅠ、FD Spe Ⅰ

FD SpeⅠ、FD Pst Ⅰ

FD EcoR Ⅰ、FD Xba Ⅰ

The enzymes using in the experiments are all from Thermo company.

5. Ligation system reaction

1)Prepare the following reaction system:

2)Incubate it more than 1 hour at 22 ℃ water bath

7. Agarose Electrophoresis

1)Prepare a piece of 1% weight-to-volume agarose and add SYBR dye or ethidium bromide

2)Place the agarose in the apparatus rig with the wells facing the negative electrode (black-colored)

3)Fill the rig with 1x TAE buffer

4)Load 5 uL 1 kb marker

5)Add 1 uL of 6x buffer to 5 uL DNA sample. Load the mixture

6)Run at 120V

8. Purification of DNA

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 gel lysis buffer to every 1 volume of gel(100mg = 100 uL)

3)Wait 2 minutes

4)Centrifuge it at 12,000 rpm for 1 minute

5)Discard the flow-through and repeat Step 4 until all sample has passed through the column

6)Add 700 uL rinse buffer into the column, then centrifuge it at 12,000 rpm for 1 minute

7)Discard the flow-through and add 500 uL rinse buffer into the column, then centrifuge it at 12,000 rpm for 1 minute

8)Discard the flow-through and centrifuge the column at 12,000 for 2 minutes

9)Transfer the column to a new centrifuge tube. Open the tube at 37℃ for a few minutes to volatilize the alcohol

10)Add 40 uL elution buffer to the center of the column and wait at least 2 minutes

11)Centrifuge it at 12,000 rpm for 1 minutes