Phase 1: PCR Amplification of Genes
From 2006.igem.org
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| - | '''A. Isolating genes for the quorum sensing mechanism from genomic ''B. subtilis'' DNA''' | + | '''A. Isolating necessary genes for the chemotaxis mechanism from genomic ''E. coli'' DNA''' |
| + | #Tsr: 1.66kb, MCP I serine receptor | ||
| + | ::*Primers: | ||
| + | :::Fwd = CGGAATTCGCTCTAGATGTTAAAACGTATCAAAATTGTGACCAGC (Tm = 70.19 deg F) | ||
| + | :::Rev = GACTGCAGCTACTAGTTAAAATGTTTCCCAGTTCTCCTCG (Tm = 70.85 F) | ||
| + | ::*Notes: | ||
| + | :::Relatively easy to obtain | ||
| + | |||
| + | |||
| + | '''B. Isolating genes for the quorum sensing mechanism from genomic ''B. subtilis'' DNA''' | ||
#ComQX: ComQ = 900bp, required accesory protein for ComX production. ComX = 168bp, pheromone | #ComQX: ComQ = 900bp, required accesory protein for ComX production. ComX = 168bp, pheromone | ||
::*Primers: | ::*Primers: | ||
| Line 8: | Line 17: | ||
:::Amplification of ComX alone was unsuccessful. However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together | :::Amplification of ComX alone was unsuccessful. However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together | ||
:::Series of DNA gel purifications (Xymo extraction) and PCR amplifications necessary to obtain sufficient quantity. | :::Series of DNA gel purifications (Xymo extraction) and PCR amplifications necessary to obtain sufficient quantity. | ||
| + | |||
| + | |||
| + | #ComP: 2.3kb, B. subtilis membrane receptor for ComX pheromone | ||
| + | ::*Primers: | ||
| + | :::Fwd = CGGAATTCCCTCTAGATGAAGAACTTAATAAAAAAATTCACAATAGCTG (Tm = 68.08 deg F) | ||
| + | :::Rev = GACTGCAGCTACTAGTTACAATTCGATTTCAATATCAGCCTTAAAGCC (Tm = 70.71 F) | ||
| + | ::*Notes: | ||
| + | :::Difficult to obtain sufficient quantity. DNA concentration by Xymo extraction following PCR amplifications, then purification from gel was necessary to obtain sufficient quantity. | ||
| + | |||
| + | #ComA: 645kb, transcription factor activated by a ligand-bound ComP. | ||
| + | ::*Primers: | ||
| + | :::Fwd = CGGAATTCCCTCTAGATGAAAAAGATACTAGTGATTGATGACCATCCGG (Tm = 72.27 F) | ||
| + | :::Rev = GACTGCAGCTACTAGTTAAAGTACACCGTCTGATTTCGCAATC (Tm = 71.39 F) | ||
| + | ::*Notes: | ||
| + | :::Relatively easy to obtain | ||
Revision as of 20:22, 26 October 2006
A. Isolating necessary genes for the chemotaxis mechanism from genomic E. coli DNA
- Tsr: 1.66kb, MCP I serine receptor
- Primers:
- Fwd = CGGAATTCGCTCTAGATGTTAAAACGTATCAAAATTGTGACCAGC (Tm = 70.19 deg F)
- Rev = GACTGCAGCTACTAGTTAAAATGTTTCCCAGTTCTCCTCG (Tm = 70.85 F)
- Notes:
- Relatively easy to obtain
B. Isolating genes for the quorum sensing mechanism from genomic B. subtilis DNA
- ComQX: ComQ = 900bp, required accesory protein for ComX production. ComX = 168bp, pheromone
- Primers:
- Fwd = CGGAATTCCCTCTAGATGAAGGAGATTGTGGAGCAAACATATTTAACG
- Rev = GACTGCAGCTACTAGTTAATCACCCCATTGACGGGTTATTGG
- Notes:
- Forward primer contains EcoRI and XbaI cut sites; Reverse primer contains SpeI and PstI cut sites
- Amplification of ComX alone was unsuccessful. However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together
- Series of DNA gel purifications (Xymo extraction) and PCR amplifications necessary to obtain sufficient quantity.
- ComP: 2.3kb, B. subtilis membrane receptor for ComX pheromone
- Primers:
- Fwd = CGGAATTCCCTCTAGATGAAGAACTTAATAAAAAAATTCACAATAGCTG (Tm = 68.08 deg F)
- Rev = GACTGCAGCTACTAGTTACAATTCGATTTCAATATCAGCCTTAAAGCC (Tm = 70.71 F)
- Notes:
- Difficult to obtain sufficient quantity. DNA concentration by Xymo extraction following PCR amplifications, then purification from gel was necessary to obtain sufficient quantity.
- ComA: 645kb, transcription factor activated by a ligand-bound ComP.
- Primers:
- Fwd = CGGAATTCCCTCTAGATGAAAAAGATACTAGTGATTGATGACCATCCGG (Tm = 72.27 F)
- Rev = GACTGCAGCTACTAGTTAAAGTACACCGTCTGATTTCGCAATC (Tm = 71.39 F)
- Notes:
- Relatively easy to obtain
