Difference between revisions of "E coli colony PCR"

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This is a protocol to do PCR reactions on ''E. coli'' cells, directly after a [[E_coli_Chemical_Transformations | transformation]]. (See [[Pombe_Colony_PCR | here for colony PCR for yeast cells]])<BR>
 
This is a protocol to do PCR reactions on ''E. coli'' cells, directly after a [[E_coli_Chemical_Transformations | transformation]]. (See [[Pombe_Colony_PCR | here for colony PCR for yeast cells]])<BR>
Prepare mastermix, divide 20μl/sample over PCR reaction tubes/wells and add some fresh cells to the mix using pipette tips or toothpicks.<BR>
+
Prepare mastermix, divide 19μl/sample over PCR reaction tubes/wells and add some fresh cells to the mix using pipette tips or toothpicks.<BR>
 
If you actually want to use these cells later, '''don't forget to maintain the cells''' on a fresh 'master plate' or in a 96 well plate. <BR>
 
If you actually want to use these cells later, '''don't forget to maintain the cells''' on a fresh 'master plate' or in a 96 well plate. <BR>
  
Make sure you add only a few cells. Adding too much might reduce the efficiency of the reaction, resulting in no product. If you're reaction fails, a solution might be to first resuspend your cells in 10μl DNAse free water and then add 1μl of this mix to the reaction.
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Re-suspend cells for each colony in 50μl DNAse free water and then add 1μl of this mix to the reaction.
  
 
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Run using program (bart -> colony PCR)
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Run using program (bart -> colony PCR or cPCR)
 
{| class="wikitable"
 
{| class="wikitable"
 
!Number of cycles  
 
!Number of cycles  

Revision as of 09:04, 11 September 2019


This is a protocol to do PCR reactions on E. coli cells, directly after a transformation. (See here for colony PCR for yeast cells)
Prepare mastermix, divide 19μl/sample over PCR reaction tubes/wells and add some fresh cells to the mix using pipette tips or toothpicks.
If you actually want to use these cells later, don't forget to maintain the cells on a fresh 'master plate' or in a 96 well plate.

Re-suspend cells for each colony in 50μl DNAse free water and then add 1μl of this mix to the reaction.

Do sequencing reaction:
H2O 15.4 μl
Taq PCR Buffer 10X 2 μl
dNTPs (10 mM each) 0.8 μl
MgCl2 (50mM) 0.8 μl
Primer Fwd (10 μM) 0.4 μl
Primer Rev (10 μM) 0.4 μl
Taq polymerase (5U/μl) 0.2 μl

Run using program (bart -> colony PCR or cPCR)

Number of cycles Temperature Duration
1 X 94°C 2 min
35X 94°C 30 sec
52°C 30 sec
72°C 1 min 30 sec
1X 72°C 7 min
7°C Inf