Silver: Verifying Genomic Integration: Difference between revisions
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== Verifying Genomic Integration == | == Verifying Genomic Integration == | ||
*Single colony PCR is used to identify transformants which have the gene of interest integrated at the genomic locus. One set of PCR primers is designed to identify whether the prototrophic or auxotrophic allele is at the genomic locus. In this case, one primer binds to the genomic DNA and the second primer binds to the coding sequence of the auxotrophic gene. The second set of PCR primers is designed to identify whether the | *Single colony PCR is used to identify transformants which have the gene of interest integrated at the genomic locus. One set of PCR primers is designed to identify whether the prototrophic or auxotrophic allele is at the genomic locus. In this case, one primer binds to the genomic DNA and the second primer binds to the coding sequence of the auxotrophic gene. The second set of PCR primers is designed to identify whether the Sikorski vector is at the genomic locus. In this case, one primer binds to the genomic DNA and the second primer binds to the Sikorski vector. | ||
* Typically, 3 transformants are screened per desired strain. Re-streak each colony onto the appropriate selection plate. | * Typically, 3 transformants are screened per desired strain. Re-streak each colony onto the appropriate selection plate. | ||
* Pick a colony at least 2 mm in diameter using a wooden toothpick. Resuspend it into 11 uL Lyse-N-Go (Pierce) in a thick-walled PCR tube. Transfer 5.5 uL of the mixture into a second PCR tube. | * Pick a colony at least 2 mm in diameter using a wooden toothpick. Resuspend it into 11 uL Lyse-N-Go (Pierce) in a thick-walled PCR tube. Transfer 5.5 uL of the mixture into a second PCR tube. | ||
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**65 C for 1 min | **65 C for 1 min | ||
**80 C forever | **80 C forever | ||
* Add 45 uL PCR mix to each tube. For each colony, you will have two PCR reactions corresponding to the two sets of PCR primers. The PCR mix is composed of: | * DO NOT remove the tubes from the pcr block for the next step. | ||
* Add 45 uL PCR mix to each tube, while in the pcr block. Pipet up and down to mix. For each colony, you will have two PCR reactions corresponding to the two sets of PCR primers. The PCR mix is composed of: | |||
** 5 uL 10x Thermopol buffer | ** 5 uL 10x Thermopol buffer | ||
** 1 uL 10 mM dNTPs | ** 1 uL 10 mM dNTPs | ||
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** 95C for 30s | ** 95C for 30s | ||
** 53C for 30s | ** 53C for 30s | ||
** 72C for | ** 72C for (URA = 1:00; TRP = 1:30, LEU = 2:00) | ||
** repeat 35 times | ** repeat 35 times | ||
** 8C forever | ** 8C forever |
Revision as of 07:24, 27 September 2006
Back to Protocols
Verifying Genomic Integration
- Single colony PCR is used to identify transformants which have the gene of interest integrated at the genomic locus. One set of PCR primers is designed to identify whether the prototrophic or auxotrophic allele is at the genomic locus. In this case, one primer binds to the genomic DNA and the second primer binds to the coding sequence of the auxotrophic gene. The second set of PCR primers is designed to identify whether the Sikorski vector is at the genomic locus. In this case, one primer binds to the genomic DNA and the second primer binds to the Sikorski vector.
- Typically, 3 transformants are screened per desired strain. Re-streak each colony onto the appropriate selection plate.
- Pick a colony at least 2 mm in diameter using a wooden toothpick. Resuspend it into 11 uL Lyse-N-Go (Pierce) in a thick-walled PCR tube. Transfer 5.5 uL of the mixture into a second PCR tube.
- Load all the tubes into the PCR machine and run the Lyse N Go PCR protocol.
- 65 C for 0.5 min
- 8 C for 0.5 min
- 65 C for 1.5 min
- 97 C for 3 min
- 8 C for 1 min
- 65 C for 3 min
- 97 C for 1 min
- 65 C for 1 min
- 80 C forever
- DO NOT remove the tubes from the pcr block for the next step.
- Add 45 uL PCR mix to each tube, while in the pcr block. Pipet up and down to mix. For each colony, you will have two PCR reactions corresponding to the two sets of PCR primers. The PCR mix is composed of:
- 5 uL 10x Thermopol buffer
- 1 uL 10 mM dNTPs
- 0.25 uL 100 uM forward primer
- 0.25 uL 100 uM reverse primer
- 0.5 uL NEB Vent polymerase
- 38 uL water (to 45 uL total)
Sikorski vector to be integrated | allele primers | vector primers |
pRS304*(TRP1) | DL11, DL17 | DL11, DL22 |
pRS305 (LEU2) | DL28, DL29 | DL28, DL30 |
pRS306 (URA3) | IP108, IP111 | IP108, DL18 |
- Run the following PCR cycle:
- 95C for 2 min
- 95C for 30s
- 53C for 30s
- 72C for (URA = 1:00; TRP = 1:30, LEU = 2:00)
- repeat 35 times
- 8C forever
- Run the PCR products on an agarose gel. Since the bands are typically rather faint, pour the gel as thick as possible, and load 35 uL PCR + dye in the 5 mm-wide lanes.
- Use the following table to determine whether the integration was successful.
gene at locus | pcr product size | |
allele primers | vector primers | |
TRP1 | 435 | 1031 |
trpD63 | none | none |
LEU2 | 1168 | 1464 |
leu2D1 | none | 979 |
URA3 | 670 | none |
ura3-52 | 699 | 651 |
Archiving Yeast Strains
- Incoculate YEP media with a single yeast colony.
- Grow overnight at 30C in a roller drum.
- Mix 50% glycerol and yeast culture in a 2 mL screw-top vial to give a final glycerol concentration of 15%(v/v), e.g. 300 uL 50% glycerol and 700 uL yeast culture.