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Team:BCCS-Bristol/Protocols-Colony PCR
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# Primers: for BioBricks the primers VF2 (forward) and VR (reverse) are used | # Primers: for BioBricks the primers VF2 (forward) and VR (reverse) are used | ||
# Master Mix (MM): “Taq 2x Master Mix” from NEB # M0270L | # Master Mix (MM): “Taq 2x Master Mix” from NEB # M0270L | ||
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===PCR-Mix for one reaction (20 µl):=== | ===PCR-Mix for one reaction (20 µl):=== | ||
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# Mix the PCR mix and give 20 µl in each PCR reaction tube (either using a new tip or ensure that you don’t get in contact with any DNA) | # Mix the PCR mix and give 20 µl in each PCR reaction tube (either using a new tip or ensure that you don’t get in contact with any DNA) | ||
# Close the tubes properly, mix them gently with the vortexer and spin them shortly down | # Close the tubes properly, mix them gently with the vortexer and spin them shortly down | ||
| - | # Start the PCR | + | # Start the PCR |
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===PCR programme:=== | ===PCR programme:=== | ||
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|Initial denaturation||95°C|| 5 min (if it’s not a colony PCR, take only 2 min) | |Initial denaturation||95°C|| 5 min (if it’s not a colony PCR, take only 2 min) | ||
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Latest revision as of 20:40, 13 September 2008
Colony PCR
Components of the PCR Mix:
- Template DNA: Colonies of a transformation plate
- Primers: for BioBricks the primers VF2 (forward) and VR (reverse) are used
- Master Mix (MM): “Taq 2x Master Mix” from NEB # M0270L
PCR-Mix for one reaction (20 µl):
| Volume | Component |
| 10 µl | MM |
| 9.6 µl | sterile water |
| 0.2 µl | forward primer (100 µM) |
| 0.2 µl | reverse primer (100 µM) |
Prepare additionally to the number of colonies you want to test one reaction for the negative control and one reaction as reserve since it might be not enough solution in the end!
PCR instructions:
- Touch with a pipette tip the colony on the plate and try to get as much bacteria as possible
- Touch now the bottom of a PCR reaction tube and turn the tip several times
- Take this tip and make a small streak out on a plate (LB + antibiotics) and incubate the plate at 37°C during a day or overnight
- Mix primers and water
- When everything is ready, take the MM out of the fridge, add it to the primers and the water and bring the MM as soon as possible back into the fridge
- Mix the PCR mix and give 20 µl in each PCR reaction tube (either using a new tip or ensure that you don’t get in contact with any DNA)
- Close the tubes properly, mix them gently with the vortexer and spin them shortly down
- Start the PCR
PCR programme:
| Initial denaturation | 95°C | 5 min (if it’s not a colony PCR, take only 2 min) |
| Denaturation | 95°C | 30 s |
| Annealing | 55°C* | 30s |
| Extension | 68°C | 1 min/1 kb fragment length |
| Final extension | 68°C | 2 min |
* or the appropriate temperature for the primers
