Team:Amsterdam/Notebook/Protocols/ColonyPCR
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{{:Team:Amsterdam/Header}} | {{:Team:Amsterdam/Header}} | ||
=Colony PCR= | =Colony PCR= | ||
- | We used this protocol to check the new transformants. | + | We used this protocol to check if the new transformants contained the right plasmids/insert. |
==Materials== | ==Materials== | ||
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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. | ||
- | #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: | + | [[Image:Colony_PCR.jpg|600px]]<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.'' | ||
- | # | + | #Incubate the selected colonoies on the LB-agar plate ON at 37°C. |
- | #Check the PCR | + | #Check the PCR-sample sizes on agarose gel. |
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{{:Team:Amsterdam/Footer}} | {{:Team:Amsterdam/Footer}} |
Latest revision as of 00:01, 22 September 2011
Colony PCR
We used this protocol to check if the new transformants contained the right plasmids/insert.
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 |
- Make the PCR mix and pippete it in a PCR tube.
- Pick a colony from a LB-agar plate using a sterile pipette tip.
- 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.
- Redo this with all the selected colonies.
- Run the following PCR program:
Note: Adjust the elongation time according to the expected product size. 1 minute for each Kb.
- Incubate the selected colonoies on the LB-agar plate ON at 37°C.
- Check the PCR-sample sizes on agarose gel.