Brandon:Protocols/Cloning Transformation3
Jump to navigation
Jump to search
cloning and A-tailing etc.[edit]
- procedure for TA cloning and tranformation of E. Coli
Cloning into vector[edit]
- make sure you know to use TA cloning or blunt cloning vector.
- procedure for cloning with Invitrogen TA cloning kit, for TA cloning.
- make sure DNA has been qiaquick cleaned if needed
1. amount of PCR product needed. = ((200)(50))/3900 = 2.5 ng 2. reaction mixture 3 uL PCR product (qiaquick purified if needed) 1 uL ligation buffer 2 uL pCR vector 2.1 (25 ng/ul) 1 uL nuclease free water 3 uL T4 DNA ligase (used 1U/uL T4 not from kit, Kit T4 is @ 5U/uL 10 uL total 3. Incubate at RT for 30 minutes or 1 hour max. OR Incubate at 14C for 4 hours or overnight 4. Now ready for transformation into E coli.
Transformation of E. coli[edit]
- transfect into E. coli, use transfection protocol
1. Thaw one vial of One Shot TOP10 chemically competent cells for each transformation Add 1-3 uL of PCR product/ligated cloning vector from above to TOP10 cell vial (uL based on calculation), MIX GENTLY, no pipetting up and down 2. incubate vials on ice for 30 minutes 3. Heat-shock cells for 30 seconds at 42C, no shaking 4. remove from 42C and place on ice for 2 minutes 5. add 250 ul pre-warmed SOC medium to each vial 6. cap vials tightly and shake horizontally at 37C for 1 hr at 224 rpm in shaking incubator 7. spread 20-200 ul of transformations onto pre-warmed selective plates and incubate overnight at 37C. *use 2-3 different concentrations, 1:10, 50 uL, 200 uL or w/e 8. Store reamining transforamtion mix at 4C. cells can be plated next day. 9. Invert selective plates, incubate at 37 overnight. 10. Select colonies and analyze with PCR or w/e.