1134 pTXB1-Ecoli-Ligase: Difference between revisions
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Latest revision as of 15:44, 6 August 2012
Cloning of 1134 pTXB1-Ecoli-Ligase
[edit]Purpose: Cloning of 1134 pTXB1-Ecoli-Ligase
Date: July 23, 2012
Notebook:
Page:
Clone ID: 1134
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]Date: July 24, 2012
See lane 3, expected size 2017 bp
PCR using these primers and genomic DNA fromm MG 1665
Ecoli_lig-Nde1-F
atcatcCATatggaatcaatcgaacaacaac
Ecoli_lig-Xho1-R
(gaaatgctgcgtttgctgggtagc CTCGAG catgca)
tgcatgCTCGAGgctacccagcaaacgcagcatttc
Clone into pTXB1-PT-ELK (1254) with RE (Nde1 + Xho1).
See lane 8, digested insert and vector together in the same lane. Insert is missing, need to reamplify. Save vector
See lane 2, expected size 2017 bp
PCR using these primers and genomic DNA from MG 1665
Ecoli_lig-Nde1-F
atcatcCATatggaatcaatcgaacaacaac
Ecoli_lig-Xho1-R
(gaaatgctgcgtttgctgggtagc CTCGAG catgca)
tgcatgCTCGAGgctacccagcaaacgcagcatttc
Clone into pTXB1-PT-ELK (1254) with RE (Nde1 + Xho1).
Digested with Nde1 and Xho1 (see lane 2), cut out band, purify and ligate together with vector saved form earlier. Ligate over night, transform tomorrow morning
Date: August 2, 2012
Screening came back bad, started all over again
See lane 3, expected size 2017 bp
PCR using these primers and genomic DNA fromm MG 1665
Ecoli_lig-Nde1-F
atcatcCATatggaatcaatcgaacaacaac
Ecoli_lig-Xho1-R
(gaaatgctgcgtttgctgggtagc CTCGAG catgca)
tgcatgCTCGAGgctacccagcaaacgcagcatttc
Clone into pTXB1-PT-ELK (1254) with RE (Nde1 + Xho1).
Ligate together, transform
Colony Screening
[edit]Date: July 27, 2012
Digested MiniPrep with RE Nde1 and Xho1, expected size 2017 bp
See lanes 19- 24
Lane 21 (sample 3) may have insert? Send sample 3 to sequencing
Date: August 6, 2012
Digested MiniPrep with RE Nde1 and Xho1, expected size 2017 bp
No inserts
Result intepretation/Summary
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Conclusion
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