Team:Paris Saclay/Protocols/PCR for bacterial culture

From 2014.igem.org

(Difference between revisions)
(→PCR for bacterial culture)
(→PCR for bacterial culture)
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Add to final volume
Add to final volume
| width="25%" |
| width="25%" |
-
37.75μl (50μl final)
+
27.75μl (50μl final)
|-
|-
| Tp Green 5X
| Tp Green 5X
| 5X
| 5X
| 1X
| 1X
-
| 5μl
+
| 10μl
|-
|-
| dNTPs  
| dNTPs  
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| 1mM
| 1mM
| 10μM
| 10μM
-
| 2.5μl
+
| 2μl
|-
|-
| Primer B
| Primer B
| 1mM
| 1mM
| 10μM
| 10μM
-
| 2.5μl
+
| 2μl
|-
|-
-
| Template DNA: Genomic DNA
+
| Culture
| -
| -
-
| 1μl
+
| -
-
| 1μl
+
| 2μl
|-
|-
| GoTaq DNA polymerasse
| GoTaq DNA polymerasse
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| 0.25μl
| 0.25μl
| 0.25μl
| 0.25μl
-
 
+
|-
 +
| DMSO
 +
| -
 +
| -
 +
| 1.5μl
|}
|}

Revision as of 12:00, 29 July 2014

PCR for bacterial culture

1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
Component Initial concentration Final concentration Example for 50μl

H2O

-

Add to final volume

27.75μl (50μl final)

Tp Green 5X 5X 1X 10μl
dNTPs 10mM 200μM 1μl
Primer A 1mM 10μM 2μl
Primer B 1mM 10μM 2μl
Culture - - 2μl
GoTaq DNA polymerasse - 0.25μl 0.25μl
DMSO - - 1.5μl
2. Program the PCR machine according to the Table 2.
Cycle step Temperature Time Cycle

Initial denaturation

95 - 98°C

30 s - 3 min

1

Denaturation 95 - 98°C 10 - 30 s 25 - 30
Annealing variable 30 s 25 - 30
Extension 72°C 30 s - 2 min 25-30
Final extension 72°C 10 min 1
Final extension 4 - 8°C hold 1
3. Verify correct amplification by agarose gel electrophoresis.

Back to the Protocols

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_for_bacterial_culture"