1118 p8H-Klenow
Cloning of 1118 p8H-Klenow
[edit]Purpose: Cloning of 1118 p8H-Klenow
Date: July 23, 2012
Notebook:
Page:
Clone ID: 1118
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]Date: July 24, 2012
See lane 1, expected size 1817 bp
PCR using MG1665 genomic DNA as template
DNA-Pol1-Sal1-R
(cgaaaactgggatcaggcgcac GTC GAC tca cgat)
atcgtgaGTCGACGCGGCCGCCgtgcgcctgatcccagttttcg
Klenow-Nde1-F
cagtcg catatg GTGATTTCTTATGACAACTAC
Cut out band, purify, digest with RE Ndel and Sal1 with vector 1135
See lanes 4 (insert) and 5 (vector)
Lost insert, need to reamplify, save the digested vector
See lane 1, expected size 1817 bp
PCR using MG1665 genomic DNA as template
DNA-Pol1-Sal1-R
(cgaaaactgggatcaggcgcac GTC GAC tca cgat)
atcgtgaGTCGACGCGGCCGCCgtgcgcctgatcccagttttcg
Klenow-Nde1-F
cagtcg catatg GTGATTTCTTATGACAACTAC
Cut out band, purify, digest with RE Ndel and Sal1
Digested with Nde1 and Sal1 (see lane 1), 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
See lane 2, expected size 1817 bp
PCR using MG1665 genomic DNA as template
DNA-Pol1-Sal1-R
(cgaaaactgggatcaggcgcac GTC GAC tca cgat)
atcgtgaGTCGACGCGGCCGCCgtgcgcctgatcccagttttcg
Klenow-Nde1-F
cagtcg catatg GTGATTTCTTATGACAACTAC
Cut out band, purify, digest with RE Ndel and Sal1 with vector 1135
See lanes 3 (insert) and 4 (vector), cut out, ligate together, transform
Colony Screening
[edit]Date: July 27, 2012
Digested MiniPrep with RE Nde1 and Sal1, expected size 1817 bp.
Sample 4 may be correct
Date: August 6, 2012
Digested MiniPrep with RE Nde1 and Sal1, expected size 1817 bp.
No inserts present
Cloning of 1118 pET28b.Amp-Klenow
Purpose: Cloning of 1118 pET28b.Amp-Klenow
Date: Dec, 2012
Notebook:
Page:
Clone ID: 1118
Experimenters: Shanyi
Results
PCR using DH5alpha genomic DNA as template
digest 1422and 1118pcr products
screen
Result intepretation/Summary
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Conclusion
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