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Team:EPF-Lausanne/Protocols/Colony PCR
From 2011.igem.org
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* Recover the colonies: | * Recover the colonies: | ||
** Choose a few colonies that you want to analyze | ** Choose a few colonies that you want to analyze | ||
| - | ** Prepare PCR tubes with | + | ** Prepare PCR tubes with 50 ul water |
** For each colony, take it with a tip, then plate it on a new agar plate (just make a dot). You can use one plate for all the colonies tested. | ** For each colony, take it with a tip, then plate it on a new agar plate (just make a dot). You can use one plate for all the colonies tested. | ||
** Put the tip in a PCR tube and mix. | ** Put the tip in a PCR tube and mix. | ||
| Line 15: | Line 15: | ||
** reaction buffer 5x (with MgCl2): 10 ul | ** reaction buffer 5x (with MgCl2): 10 ul | ||
** dNTPs 10mM: 1 ul | ** dNTPs 10mM: 1 ul | ||
| - | ** each primer ( | + | ** each primer (20 uM): 1 ul (nneds to be 0.4 uM in final volume) |
** template DNA (boiled samples): 1ul | ** template DNA (boiled samples): 1ul | ||
** HiFi PLUS (5 U/ul): 0.5 ul | ** HiFi PLUS (5 U/ul): 0.5 ul | ||
| Line 21: | Line 21: | ||
| - | * For J61002 Ptet-RFP plasmid: | + | * For J61002 Ptet-RFP plasmid (control to see if colony PCR is working): |
** use J6-Ptet_RFP-f and J6-Ptet_RFP-r, use the "VINCECOL" file | ** use J6-Ptet_RFP-f and J6-Ptet_RFP-r, use the "VINCECOL" file | ||
* For reporter plasmid: | * For reporter plasmid: | ||
Latest revision as of 08:19, 22 August 2011
Colony PCR
Back to protocols.This step takes place after having transformed cells with Gibson-assembled plasmids and having grown them overnight on a plate. It is quicker to check the plasmids at this stage rather than waiting for a miniprep.
- Recover the colonies:
- Choose a few colonies that you want to analyze
- Prepare PCR tubes with 50 ul water
- For each colony, take it with a tip, then plate it on a new agar plate (just make a dot). You can use one plate for all the colonies tested.
- Put the tip in a PCR tube and mix.
- Boil the tubes at 95°C for 10min (use the "BOIL" file)
It is important to place the colonies on a new plate, because if the PCR gives good results we need the corresponding cells!
- Prepare the PCR: protocol adapted for HiFi PLUS enzyme, for 50 ul final volume
- reaction buffer 5x (with MgCl2): 10 ul
- dNTPs 10mM: 1 ul
- each primer (20 uM): 1 ul (nneds to be 0.4 uM in final volume)
- template DNA (boiled samples): 1ul
- HiFi PLUS (5 U/ul): 0.5 ul
- ddH20: 35.5 ul (check to have 50ul total)
- For J61002 Ptet-RFP plasmid (control to see if colony PCR is working):
- use J6-Ptet_RFP-f and J6-Ptet_RFP-r, use the "VINCECOL" file
- For reporter plasmid:
- use J6Ptet-LacI-RFPf and J6Ptet-LacI-RFPr
- For lysis plasmid:
- use J6Ptet-LacI-RFPf and J6Ptet-LacI-Lysr
