"
Team:HIT-Harbin/Protocol
From 2013.igem.org
Nootbook/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
3.Transformation
1)Thaw the competent cells on ice
2)Add 50 uL of thawed competent cells into pre-chilled 1.5ml tube
3)Add the total ligation plasmid into the 1.5 ml tube. Overturn gently the tube a few times. Make sure that the competent cells is always on ice
4)Close the tubes and incubate the mixture on ice for 30 minutes
5)Heat shock the cells by water bath at 42℃ for 45 seconds
6)Place the cells on ice for 1 minutes
7)Add 400 uL LB liquid media into the tubes
8)Incubate the cells at 37℃, 110rpm for 1 hour
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.
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
2)Incubate it more than 1 hour at 22 ℃ water bath
6. PCR reaction
1)Prepare the following reaction system:
2)The PCR reaction setting:
2)The PCR reaction setting:
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
