Team:JNFLS/Protocols

Protocols

PCR truncation of HCVC gene

Primers:

tu1,2,3

Enzyme digestion of pcoldII vector plasmid

tu4

  1. Enzyme digestion of pcoldII vector plasmid
  2. add 5ul green buffer
  3. add 1ul Xhol
  4. add 1ul PstI
  5. add distilled water to 20μL
  6. water bath at 37 degrees for 10 min

Ligation

tu5

Bacterial transformation

  1. Remove cells from -80°C freezer and thaw in hand
  2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex
  3. Place the mixture on ice for 2 minutes. Do not mix
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 2 minutes. Do not mix.
  6. Pipette 250 μl of room temperature SOC into the mixture. Immediately spread 50-100 μl onto a selection plate and incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs may be unstable at elevated temperatures.

Colony PCR

tu6

Conditions：

tu7

Gel Recycling

  1. Run electrophoresis separation of the target DNA fragment
  2. Cut off the target DNA fragments, as far as possible the unwanted cut off
  3. The cut of the glue into the 1.5m | plastic centrifuge tube, said the quality of the plastic
  4. Add the same amount of Binding Buffer(XP2), that is, add 0.3m| liquid if 0.3g is weighed
  5. Heat in a 50-60 degree bath for a few minutes until all the glue is melted, stirring to mix.
  6. Put the HiBind DNA Mini Column into the 2ml collection tube
  7. Add the HiBindDNA Mini Column with less than 700ul from the DNA solution in step 5
  8. Centrifuge 60s at 12000rpm at room temperature
  9. Discard the waste liquid and recycle the collection pipe
  10. Repeat steps 7-9 until all samples are transferred to column
  11. Add 300ul Binding Buffer
  12. Centrifuge 60s at 12000rpm at room temperature
  13. Discard the waste liquid and recycle the collection pipe
  14. Add the 700ul SPW Wash Buffer
  15. Centrifuge 60s at 12000rpm at room temperature
  16. Discard the waste liquid and recycle the collection pipe once again in steps 14-16
  17. Centrifuge the empty HiBind DNA Mini Column 1 2000rmp for 2 minutes
  18. The Hibind DNA Mini Column was transferred to 1.5m| plastic centrifuge tube
  19. Add 15-30ul Elution Buffer to the center of the membrane
  20. Let sit at room temperature for 2 minutes
  21. Centrifugation for 12000rmp at room temperature for 1 minute

plasmid extraction from positive clones using an extraction kit

  1. Decant or aspirate and discard the culture media.Centrifuge at 10,000x g for 1 minute at room temperature
  2. Add 250 uL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.Complete resuspension of cell pellet is vital for obtaining good yields.
  3. Transfer suspension into a new 15 mL microcentrifuge tube.
  4. Add250 uL Solution IL Invert and gently rotate the tube several times to obtain a dear lysate. A 2-3minute incubation may be necessary.
  5. Add 350uL Solution I Immediately invert several times until a flocculent white precipitate form.
  6. Centrifuge at maximum speed (213,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
  7. Insert a HiBinde DNA Mini Column into a 2 mL Collection Tube.
  8. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind* DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind" DNA Mini Column.
  9. Centrifuge at maximum speed for 1 minute.
  10. Discard the filtrate and reuse the collection tube.
  11. Add 500 uL HBC Buffer
  12. centrifuge at maximum speed for 1 minute.
  13. Discard the filtrate and reuse collection tube.
  14. Add 700uL DNA Wash buffer.
  15. Centrifuge at maximum speed for 1 minute.
  16. Discard the filtrate and reuse the collection tube.
  17. Centrifuge the empty HiBind" DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
  18. Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
  19. Add 30100 uL Elution Buffer or sterile deionized water directly to the center of the column membrane.
  20. Let sit at room temperature for 1 minute.
  21. Centrifuge at maximum speed for 1 minute. Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration
  22. Store DNA at -20degrees.

Purification of recombinant protein with His tag