Team:Amsterdam/Notebook/Protocols/ColonyPCR

From 2011.igem.org

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#Make the PCR mix and pippete it in a PCR tube.
#Make the PCR mix and pippete it in a PCR tube.
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#Pick a colony from a LB-agar plate using a sterile pipette tip
+
#Pick a colony from a LB-agar plate using a sterile pipette tip.
-
#Pippete up and down in the PCR mix
+
#Pippete up and down in the PCR mix.
#Cross the pippete tip on the new LB-agar plate in the box corresponding to the sample number.
#Cross the pippete tip on the new LB-agar plate in the box corresponding to the sample number.
-
#Redo this with all the selected colonies
+
#Redo this with all the selected colonies.
-
#Run the following PCR program
+
#Run the following PCR program:
[[Image:Colony_PCR.jpg]]<br>
[[Image:Colony_PCR.jpg]]<br>
''Note: Adjust the elongation time according to the expected product size. 1 minute for each Kb.''
''Note: Adjust the elongation time according to the expected product size. 1 minute for each Kb.''
-
#Place the LB-agar plate with the selected colonies ON at 37&deg;C
+
#Incubate the selected colonoies on the LB-agar plate ON at 37&deg;C.
-
#Check the PCR samples on agarose gel
+
#Check the PCR-sample sizes on agarose gel.

Revision as of 11:53, 19 September 2011

Colony PCR

We used this protocol to check the new transformants.

Materials

  • Petri dish with LB agar and appropriate antibiotic
  • ON colonies on a petri dish with LB agar and appropriate antibiotic
  • dNTPs
  • primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VR])
  • Taq polymerase
  • Taq buffer
  • dH2O
  • PCR tubes
  • PCR machine

Protocol

Sample µl
VF2 primer 0,5
VR primer 0,5
dNTPs 1
Taq buffer 2,5
Taq polymerase 0,2
H2O 20,3
Total 25 µl

  1. Make the PCR mix and pippete it in a PCR tube.
  2. Pick a colony from a LB-agar plate using a sterile pipette tip.
  3. Pippete up and down in the PCR mix.
  4. Cross the pippete tip on the new LB-agar plate in the box corresponding to the sample number.
  5. Redo this with all the selected colonies.
  6. Run the following PCR program:

Colony PCR.jpg
Note: Adjust the elongation time according to the expected product size. 1 minute for each Kb.

  1. Incubate the selected colonoies on the LB-agar plate ON at 37°C.
  2. Check the PCR-sample sizes on agarose gel.





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