Difference between revisions of "E coli colony PCR"
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{{DISPLAYTITLE:''E coli'' colony PCR}} | {{DISPLAYTITLE:''E coli'' colony PCR}} | ||
− | 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]]) | + | 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]]) |
− | Prepare mastermix | + | |
− | + | * Prepare mastermix | |
+ | |||
+ | * Divide 19μl/sample over PCR reaction tubes/wells | ||
+ | * Prepare cells in water | ||
+ | * Add cell mixture to the mix | ||
Re-suspend cells for each colony in 50μl DNAse free water and then add 1μl of this mix to the reaction. | Re-suspend cells for each colony in 50μl DNAse free water and then add 1μl of this mix to the reaction. |
Revision as of 20:42, 3 January 2021
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
- Prepare cells in water
- Add cell mixture to the mix
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 |