Objectives
To obtain DNA of human CASPR1 and human CNTN1 without tags.
Materials
- DH5alpha competent cells
- CASPR1: EX-M4
- CNTN1: EX-M
- LB (25g per litre)
- Bacterial plates: LB+agar+ampicillin
Methods
- Thaw competent cells on ice and chill 2 14ml Falcon tubes
- Put 100ul of competent cells in each tube
- Incubate them on ice for 30 min
- Heat-shock cells for 45 seconds at 42ºC
- Incubate them 2 minutes on ice
- Add 0.9ml of SOC medium
- Incubate it 1h at 37ºC 225rpm
- Centrifugate the cultured bacterial cells at 6000G for 15 min and discard the supernatant
- Place a Plasmid Maxi Column in a 50ml centrifuge tube
- Equilibrate it by adding 10ml of PEQ buffer, allow the columns to empty by gravity flow
- Discard the filtrate and place the column back in the tube
- Add 10 ml of PM1 buffer to resuspend the cell pellet completely
- Add 10ml of PM2 buffer and mix gently by inverting the tube 10 times (do not vortex)
- Let stand at RT for 2 minutes
- Add 10ml of PM3 and mix immediately by shaking the tube vigorously for 10seconds
- Centrifuge 6000G for 20min at RT
- Transfer the supernatant to the column and allow it to flow through by gravity flow
- Discard the filtrate and place the column back in the tube
- Wash the column by adding 30ml of PW buffer, allow the column to empty by gravity flow
- Discard the filtrate
- Place the column in a clean tube and add 12ml of PEL buffer to elute the DNA by gravity flow
- Precipitate the DNA by adding 9ml of isopropanol to the aluted DNA
- Mix the tube completely and then centrifuge at 15000G for 30min at 4ºC
- Carefully remove the supernatant and wash the DNA pellet with 5ml of 75% ethanol
- Centrifuge at 15000G dor 10min at 4ºC
- Carefully remove the supernatant and air-dry the DNA pellet for 10min
- Dissolve the DNA pellet in 500ul of H2O
- Measure in the nanodrop the concentration
Results
- CASPR1-Human-Genecopoeia: 1.4ug/ul
- CNTN1-Human-Genecopoeia: 1.8ug/ul
