Team:TzuChiU Formosa/Protocol

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Competent cell

1. Culture DH5-alpha in a glass test tube with 3CC LB at 37°C for 12 hours.
2. Add 200 ul bacterium into a 500cc conical flask with 50 ml LB inside. Culture at 37°C until OD600 reaches 0.2
3. 3. Distribute the bacterium from the conical flask into a 50cc Centrifuge and centrifuge at 4000rpm, 15 minutes at 4 °C
4. Remove supernatant and add in 16 ml CaCl2 (100mM)
5. Centrifuge at 4000rpm, 15 minutes at 4 °C
6. Remove supernatant and add 3 ml CaCl2 (100mM)
7. Centrifuge at 4000rpm, 15 minutes at 4 °C
8. Remove supernatant and add 3 ml CaCl2 (100mM)
9. Place on ice and put in 4 °C cold room for an hour.
10. Cut tip and add 1 ml Glycerol (Takes up 25% of the total volume)
11. 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.
12. Plating the next day to test CFU (colony frequency unit)

Competent CFU test - Transformation-E.coli

Test competent cell CFU the day before

1. Defrost the 200 ul competent cell on ice.
2. Add 10 ng plasmid into the 200 ul competent cell.
3. Place it on ice for 30 minutes
4. Heat shock in water bath at 42℃ for 1 and a half minute then place it on ice for 5 minutes.
5. Add in 800 ul LB and place it in the 37℃ incubator for 1 hour.
6. Test the frequency
7. Incubate at 37℃for 12~16 hours
8. Select colony and check via quick- screening.

Glycerol Stock

Stock
1.8 % NaCl
50 % glycerol

1. Transfer 500 µl Bacterium (with LB) into freezing tube.
2. Add 500 µl stock (Bacterium::Stock = 1:1)
3. Store at -80℃

   *Rest of the bacterium should be autoclaved and disposed.

Digestion

1. Do a nanodrop test on the plasmid we want to cleave.
2. 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.

Ligation

1. 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