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






Gel Extraction Kit
  1. Excise the gel slice containing the DNA band with a clean, sharp scalpel.
  2. Weigh the gel slice. Add 1–2 volumes of diffusion buffer to 1 volume of gel.
  3. Incubate at 65°C for 10 min.
  4. Centrifuge the sample for 1 min.
  5. Carefully remove the supernatant using a pipet or a drawn-out Pasteur pipet. Pass the supernatant through a disposable plastic column or a syringe containing either a Whatman GF/C filter or packed, siliconized glass wool to remove any residual polyacrylamide.
  6. Determine the volume of the recovered supernatant.
  7. Add 3 volumes of Buffer QG to 1 volume of supernatant and mix. Check that the color of the mixture is yellow.
  8. Place a QIAquick Spin Column in a provided 2 ml collection tube.
  9. To bind DNA, apply the sample to the QIAquick Spin Column and centrifuge for 1 min.
  10. Discard flow-through and place QIAquick Spin Column back into the same collection tube.
  11. To wash, add 0.75 ml Buffer PE to column and centrifuge for 1 min.
  12. Discard flow-through and place QIAquick Spin Column back in the same tube.Centrifuge column for an additional 1 min at maximum speed.
  13. Place QIAquick Spin Column into a clean 1.5 ml microcentrifuge tube.
  14. To elute DNA, add 50 μl TE Buffer or water to the center of the QIAquick Spin Column and centrifuge for 1 min.

Plasmid Purification
  1. Pellet of the cells by centrifugation for 10 min at 12000rpm.
  2. Pour off the supernatant and remove excess media.
  3. Completely resuspend the cell pellet in 100 μl of Solution I by pipetting or vortex.
  4. Add 200 μl freshly prepared Solution II(2% SDS:0.4N NaOH = 1:1)and mix by inverting the tube 5-7 times, then let stand for 5 min.
  5. Add 150 μl Solution III and mix by inverting the tube ten times, put on ice for 5 min, then centrifuge at 12000rpm, 10 min at 4℃.
  6. Transfer the supernatant into a new eppendorf.
  7. Add 3 μl RNaseA, and put in 37℃ water bath for 30 min.
  8. Add 1 volume phenol/chloroform, inverting gently.
  9. Centrifuge at 12000rpm, 10 min at 4℃.
  10. Transfer the supernatant into a new eppendorf.
  11. Add 1 volume chloroform, inverting gently.
  12. Centrifuge at 12000rpm, 10 min at 4℃.
  13. Transfer the supernatant into a new eppendorf.
  14. Add 1 volume 95% ethanol, and put it on room temperature for 20 min.
  15. Centrifuge at 12000rpm, 10 min at 4℃.
  16. Air dry the pellet for 30 min or overnight.
  17. Add 30 μl TE buffer to elute.




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