Team:TzuChiU Formosa/Protocol
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Competent cell
- Culture DH5-alpha in a glass test tube with 3CC LB at 37°C for 12 hours.
- Add 200 ul bacterium into a 500cc conical flask with 50 ml LB inside. Culture at 37°C until OD600 reaches 0.2
- 3. Distribute the bacterium from the conical flask into a 50cc Centrifuge and centrifuge at 4000rpm, 15 minutes at 4 °C
- Remove supernatant and add in 16 ml CaCl2 (100mM)
- Centrifuge at 4000rpm, 15 minutes at 4 °C
- Remove supernatant and add 3 ml CaCl2 (100mM)
- Centrifuge at 4000rpm, 15 minutes at 4 °C
- Remove supernatant and add 3 ml CaCl2 (100mM)
- Place on ice and put in 4 °C cold room for an hour.
- Cut tip and add 1 ml Glycerol (Takes up 25% of the total volume)
- Transfer 200 ul into each eppendorf ( This step should be done quickly and taken to -80°C ASAP)
* Can use dry ice or ice with alcohol.
- Plating the next day to test CFU (colony frequency unit)
* Can use dry ice or ice with alcohol.
Competent CFU test - Transformation-E.coli
- Test competent cell CFU the day before
- Defrost the 200 ul competent cell on ice.
- Add 10 ng plasmid into the 200 ul competent cell.
- Place it on ice for 30 minutes
- Heat shock in water bath at 42℃ for 1 and a half minute then place it on ice for 5 minutes.
- Add in 800 ul LB and place it in the 37℃ incubator for 1 hour.
- Test the frequency
- Incubate at 37℃for 12~16 hours
- Select colony and check via quick- screening.
Glycerol Stock
- Stock
1.8 % NaCl
50 % glycerol
- Transfer 500 µl Bacterium (with LB) into freezing tube.
- Add 500 µl stock (Bacterium::Stock = 1:1)
- Store at -80℃
- *Rest of the bacterium should be autoclaved and disposed.
1.8 % NaCl
50 % glycerol
- *Rest of the bacterium should be autoclaved and disposed.
Digestion
- Do a nanodrop test on the plasmid we want to cleave.
- Add in the materials as follows:
- Plasmid or DNA X ul (Total of 10ug therefore add approximately >5ul)
10x buffer 10 ul
RE 1 u ( 1 ul per restriction enzyme)
dd water add to 100 ul
total 100 ul
Let in react in 37°C water bath.
*An electrophoresis can be done to double check if we have successfully cleaved our plasmid. Only one band should be seen.
- Plasmid or DNA X ul (Total of 10ug therefore add approximately >5ul)
10x buffer 10 ul
RE 1 u ( 1 ul per restriction enzyme)
dd water add to 100 ul
total 100 ul
*An electrophoresis can be done to double check if we have successfully cleaved our plasmid. Only one band should be seen.
Ligation
- Pepare cocktail
-
-
Insert DNA
-
5µl
-
plasmid
-
1µl
-
10X buffer
-
1µl
-
ligase
-
1µl
-
10mM ATP
-
1µl
-
ddH2O
-
1µl
-
total
-
10µl
- Place it in 22℃ dry bath for 1hr/overnight
- * Insert DNA : Plasmid = M ole ratio 5:1 or 3:1
- Pepare cocktail
-
- Insert DNA
- 5µl
- plasmid
- 1µl
- 10X buffer
- 1µl
- ligase
- 1µl
- 10mM ATP
- 1µl
- ddH2O
- 1µl
- total
- 10µl
-
![](https://static.igem.org/mediawiki/2013/d/dc/Protocol.png)