Melbourne/Colony PCRl
From 2007.igem.org
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# Take 3ul of resuspension and make an index plate. | # Take 3ul of resuspension and make an index plate. | ||
# 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:<BR> 1. 10ul supernatant<BR>2. 1ul forward primer<BR>3. 1ul reverse primer<BR>4. 12 ul GoTaq |
| - | + | # Use a PCR machine with the following cycles:<BR>1. 95C 10min<BR>2. (92C 1 min 54C 1min 72C 1min) X 30<BR>3. 72C 10min<BR>4. 4C hold. | |
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| - | # Use a PCR machine with the following cycles: | + | |
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# [[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 11:43, 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:
1. 10ul supernatant
2. 1ul forward primer
3. 1ul reverse primer
4. 12 ul GoTaq - Use a PCR machine with the following cycles:
1. 95C 10min
2. (92C 1 min 54C 1min 72C 1min) X 30
3. 72C 10min
4. 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
