20140416/2 Maxi prep NF155 and CNTN1-GFP

Objective

Amplify DNA to transfect HEK (CNTN-GFP and NF155)

Materials

• One Shoot TOP10 Chemically competent E. coli transfected with NF155 (pCMV6-NF155-DDKMyc (Human))
• One Shoot TOP10 Chemically competent E. coli transfected with CNTN-GFP (pEGFP-N2-CNTN1/F1 (Marsella))
• LB
• kanamycin
• QIAfilter Plasmid Midi and Maxi kits
• Nanodrop

Procedure

1. Select one isolated colony and take it off from the plate and incubate it with 4ml LB + kanamycin for approximately 4-5 hours at 37ºC in agitation (225rpm)
2. Put the culture in an Erlenmeyer with 200ml LB+kanamycin and incubate it o/n at 37ºC in agitation (225rpm)
3. Centrifuge the culture at 6000G for 15 minutes at 4ºC
4. Completely resuspend pellet in 10 ml Buffer P1
5. Add 10ml Buffer P2, mix by inverting and incubate until the solution turn blue (5 min)
6. During the incubation, screw the cap onto the outlet nozzle of the QIAfilter Cartridge
7. Add 10ml Buffer P3 to the lysate, and mix immediately and thoroughly by inverting until it is completely colorless
8. Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 minutes
9. Equilibrate a HiSpeed Tip with 10 ml Buffer QBT, allowing it to enter the resin
10. Remove the cap from the QIAfilter Cartridge outlet nozzle. Gently insert the plunger into he QIAfilter Cartridge, and filter the cell lysate into the equilibrated HiSpeed Tip
11. After the lysate has entered, wash the HiSpeed Tip with 60ml Buffer QC
12. Elute DNA with 15ml Buffer QF
13. Precipitate DNA by adding 10.5ml isopropanol, mix, and incubate for 5 min
14. During the incubation, remove the plunger from a 30ml syringe and attach the QIAprecipitator Module onto the outlet nozzle
15. Place the QIAprecipitator over a waste bottle, transfer the eluate-isopropanol mixture into the syringe, and insert the plunger. Filter the eluate-isopropanol mixture through the QIAprecipitator using constant pressure.
16. Remove the QIAprecipitator from the syringe and pull out the plunger. Re-attach the QIAprecipitator and add 2ml 70% ethanol to the syringe. Wash the DNA by inserting the plunger and pressing the ethanol through the QIAprecipitator.
17. Remove the QIAprecipitator from the syringe and pull out the plunger. Attach the QIAprecipitator again, insert the plunger, and dry the membrane by pressing air through the QIAprecipitator forcefully. Repeat this step several times
18. Dry the outlet nozzle of the QIAprecipitator with adsorbent paper
19. Remove the plunger from a new 5ml syringe, attach the QIAprecipitator and hold the outlet over a 1.5ml collection tube. Add 1ml Buffer TE to the 5 ml syringe. Insert the plunger and elute the DNA into a collection tube using constant pressure
20. Remove the QIAprecipitator from the 5 ml syringe, pull out the plunger and re-attach the QIAprecipitator to the 5ml syringe
21. Tranfer the eluate from step 19 to the 5 ml syringe ans eluate for a second time into the same 1.5ml tube.
22. Mesure the DNA with nanodrop

Results & conclusions

We obtained 1037.5 ng/ul of NF155; and 1112.5 ng/ul of CNTN-GFP.

Aliquotes were stored at -80ºC (one aliquote at 4ºC, maxis in use).