Brandon:Protocols/Cloning Transformation3

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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.