1037 p8H-T7ligase
Cloning of 1037 p8H-T7ligase into p8H-T7ligase
[edit]Purpose: Cloning of 1037 p8H-T7ligase into p8H-T7ligase
Date: 06/04/2012
Notebook: Jennifer Truong
Page: 003347-003349
Clone ID: 1037
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
6/5/2012: No growth on plates, redid from digestion
Digested with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night
06/06/2012: No growth on plates, redid PCR product, no product, do experiment again, maybe synthesized template incorrectly or PHUSION enzyme not efficient?
Redid amplication, cut out band, purified it for digestion
Digested with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate 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 with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night
Colony Screening
[edit]Date: 06082012
All samples (1-7) were positive, grew samples 1-6 in KAN media overnight (lanes 12-17)
Prepared MiniPrep on samples, digested with enzymes to check for inserts, submitted sample 3 (lane 9) for sequencing.
Result intepretation/Summary
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