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Team:USTC-China/Protocol
From 2011.igem.org
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| + | *Aptamer-cheZ Device Construction | ||
| + | <p>The cheZ gene was cloned from E.coli TOP10 strain using PCR. A cassete containing a theophylline-sensitive aptamer, and the cheZ gene was assembled using PCR and was subcloned into the SpeI and PstI sites of pSB1A2 containing the lac promoter.</p> | ||
| + | <p>Clone Primer:</p> | ||
| + | Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGATGCAACCATCAATCAAACC-3' | ||
| + | Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGTCTATCCAACAAATCGT-3' | ||
| + | <p>Assemble Primer:</p> | ||
| + | <p>Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGGGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACCCCGCTGCAAGACAACAAG ATGCAACCATCAATCAAACC-3'</p> | ||
| + | <p>Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGACTATCCAACAAATCGT-3'</p> | ||
| + | <p>Assemble condition: 96℃ 10min, (96℃ 30s, 56℃ 30s, 72℃ 1min)16 cycles and each cycle the annealing temperature increase 1℃, (96℃ 30s, 72℃ 30s, 72℃ 1min)20 cycles,72℃ 10min and holding at 4℃.</p> | ||
Revision as of 10:01, 2 October 2011
- Aptamer-cheZ Device Construction
The cheZ gene was cloned from E.coli TOP10 strain using PCR. A cassete containing a theophylline-sensitive aptamer, and the cheZ gene was assembled using PCR and was subcloned into the SpeI and PstI sites of pSB1A2 containing the lac promoter.
Clone Primer:
Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGATGCAACCATCAATCAAACC-3' Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGTCTATCCAACAAATCGT-3'
Assemble Primer:
Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGGGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACCCCGCTGCAAGACAACAAG ATGCAACCATCAATCAAACC-3'
Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGACTATCCAACAAATCGT-3'
Assemble condition: 96℃ 10min, (96℃ 30s, 56℃ 30s, 72℃ 1min)16 cycles and each cycle the annealing temperature increase 1℃, (96℃ 30s, 72℃ 30s, 72℃ 1min)20 cycles,72℃ 10min and holding at 4℃.
