Melbourne/Colony PCRl
From 2007.igem.org
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# Take 30ul aliquot and boil at 95degrees C for 10 minutes | # Take 30ul aliquot and boil at 95degrees C for 10 minutes | ||
# Use 10ul of resultant supernatant as template in PCR reaction: | # Use 10ul of resultant supernatant as template in PCR reaction: | ||
| - | * | + | *10ul supernatant |
| - | * | + | *1ul forward primer |
| - | * | + | *1ul reverse primer |
| - | * | + | *12 ul GoTaq |
# Use a PCR machine with the following cycles: | # Use a PCR machine with the following cycles: | ||
| - | * | + | *95C 10min |
| - | * | + | *(92C 1 min 54C 1min 72C 1min) X 30 |
| - | * | + | *72C 10min |
| - | * | + | *4C hold. |
# [[Melbourne/Loading a DNA gel|Run]] the PCR products on a gel. | # [[Melbourne/Loading a DNA gel|Run]] the PCR products on a gel. | ||
Revision as of 11:41, 8 October 2007
<Return to list of protocols> <Team home page>
- Applications: Confirm presence of insert in bacterial plasmid
- Time to complete protocol:
- Lab time: 5
- Waiting time: 4hours
- Approximate cost of materials: $0.00
Contents |
Method from primary and secondary reagents
- Scrape a colony with a sterile pipette tip and resuspend in 50ul of milliQ water.
- Take 3ul of resuspension and make an index plate.
- Take 30ul aliquot and boil at 95degrees C for 10 minutes
- Use 10ul of resultant supernatant as template in PCR reaction:
- 10ul supernatant
- 1ul forward primer
- 1ul reverse primer
- 12 ul GoTaq
- Use a PCR machine with the following cycles:
- 95C 10min
- (92C 1 min 54C 1min 72C 1min) X 30
- 72C 10min
- 4C hold.
- Run the PCR products on a gel.
Primary & secondary Reagents Required including controls
- milliQ water
- GoTaq
- Forward and Reverse primers
- Bacterial colony
Equipement Required
- PCR machine
- Heating block
