Phase 1: PCR Amplification of Genes
From 2006.igem.org
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:::Rev = GACTGCAGCTACTAGTTAAAATGTTTCCCAGTTCTCCTCG (Tm = 70.85 F) | :::Rev = GACTGCAGCTACTAGTTAAAATGTTTCCCAGTTCTCCTCG (Tm = 70.85 F) | ||
::*Notes: | ::*Notes: | ||
| - | :::Relatively easy to obtain | + | :::Relatively easy to obtain. |
| - | + | :::Forward primer contains EcoRI and XbaI cut sites; Reverse primer contains SpeI and PstI cut sites. [Similar to all primers] | |
'''B. Isolating genes for the quorum sensing mechanism from genomic ''B. subtilis'' DNA''' | '''B. Isolating genes for the quorum sensing mechanism from genomic ''B. subtilis'' DNA''' | ||
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:::Rev = GACTGCAGCTACTAGTTAATCACCCCATTGACGGGTTATTGG | :::Rev = GACTGCAGCTACTAGTTAATCACCCCATTGACGGGTTATTGG | ||
::*Notes: | ::*Notes: | ||
| - | + | :::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. | ||
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:::Rev = GACTGCAGCTACTAGTTAAAGTACACCGTCTGATTTCGCAATC (Tm = 71.39 F) | :::Rev = GACTGCAGCTACTAGTTAAAGTACACCGTCTGATTTCGCAATC (Tm = 71.39 F) | ||
::*Notes: | ::*Notes: | ||
| - | :::Relatively easy to obtain | + | :::Relatively easy to obtain. |
Revision as of 20:24, 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.
- Forward primer contains EcoRI and XbaI cut sites; Reverse primer contains SpeI and PstI cut sites. [Similar to all primers]
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:
- 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.
