1143 p8H-T7-Exo-1
Cloning of 1143 p8H-T7-Exo-1 into p8H-T7ligase
[edit]Purpose: Cloning of 1143 p8H-T7-Exo-1 into p8H-T7ligase
Date: 06/04/2012
Notebook: Jennifer Truong
Page: 003347-003349
Clone ID: 1143
Experimenters: Jennifer Truong
Cloning
[edit]Date: 06/04/2012
PCR Template:
Isolated band for cloning
Digested with Nde1 ad Xho1, using p8H (1135) template
Cut out bands for ligation, transformed DH5 alpha cells and grew on KAN R plates
6/05/12: Plates showed no growth, restart from digestion
Digested with Nde1 + Xho1
Cut out bands (1143 DNA and p8H 1135 vector), purified DNA, ligated, transformed with DH5 alpha cells and grew on KAN R plates over night
06/06/2012: No growth overnight. Redid gene synthesis, cut out bands for digestion
Digested purified DNA and p8H vector with Nde1 and Xho1, cut out bands for ligation
Redid amplifaction, cut out band, purified it for digestion
Cut out bands (1143 DNA and p8H 1135 vector), purified DNA, ligated, transformed with DH5 alpha cells and grew on KAN R plates over night
06/07/2012: Again, no growth on plates.
Synthesized PCR product again, cut out circled band, purified DNA, digested DNA and vector for digestion
Digested purified DNA and p8H vector with Nde1 and Xho1, cut out bands for ligation (lanes 3 and 4)
06/08/2012: Very few colonies, amplified again with KAPA enzyme (20 cycles)
Cut out band for purification and digestion
Digested purified DNA and p8H vector with Nde1 and Xho1, cut out bands for ligation
Colony Screening
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Grew samples 1 and 2 in media (lanes 19 and 20)
Prepared MiniPrep of samples, digested with Nde1 and Xho1 to confirm inserts
Submitted sample 1 for sequencing
Result intepretation/Summary
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