Brandon:Protocols/Cloning Transformation3: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Bsos
(Created page with "==cloning and A-tailing etc.== 1. reaction from the manual transfect into E. coli, use transfection protocol 1. Thaw one vial of One Shot TOP10 chemically competent cel...")
 
>Bsos
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
==cloning and A-tailing etc.==
==cloning and A-tailing etc.==


1. reaction from the manual
*procedure for TA cloning and tranformation of E. Coli




transfect into E. coli, use transfection protocol
===Cloning into vector===


*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===
*transfect into E. coli, use transfection protocol


  1.  Thaw one vial of One Shot TOP10 chemically competent cells for each transformation
  1.  Thaw one vial of One Shot TOP10 chemically competent cells for each transformation
     Add 1-3 uL of PCR product to TOP10 cell vial (uL based on calculation)
     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
     MIX GENTLY, no pipetting up and down
   
   
Line 21: Line 43:
  6.  cap vials tightly and shake horizontally at 37C for 1 hr at 224 rpm in shaking incubator
  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.
  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
     *use 2-3 different concentrations, 1:10, 50 uL, 200 uL or w/e
   
   

Latest revision as of 21:31, 18 May 2012

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.