1137 p8H-Exo-VIII
Cloning of 1137 p8H-Exo-VIII
[edit]Purpose: Cloning of 1137 p8H-Exo-VIII
Date: June 26, 2012
Notebook:
Page:
Clone ID: 1137
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]Date: June 26, 2012
Amplified DNA with PHUSION enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature)
Cut out band (expected size = 2602 bp), purified and digested wth vector 1135 (p8H) with restriction enzymes Nde1 and Xho1
Cut out bands in lanes 1 and 2, purified and ligated together overnight.
Date: July 18, 2012
Cut out bands in lane 1 and 2 (expected size = 2602 bp), purified and digested wth vector 1135 (p8H) with restriction enzymes Nde1 and Xho1
Cut out bands in lanes 1 (insert) and 2 (vector), purified and ligated together overnight.
Colony Screening
[edit]
Screened coloines, samples 3, 4, 5, 6, 7 (?) and 8 were positive.
Grew in media overnight, do MiniPrep tomorrow
Date: June 29, 2012
Digested Mini prep (see lanes 1-6)with Nde1 + Xho1
Sent sample 3 (lane 3) to sequencing.
Date: July 12, 2012
Sequencing came back bad, grow more bacteria from stock solution, redid MiniPrep, digested plasmids with RE Xho1 and Nde1 (see lanes 5 and 6). Expected size ~2600 bp. Both samples had postive inserts, sent sample 1 (lane 5) to sequencing
Date: June 16, 2012
Sequencing came back bad, grow more bacteria from plate, redid MiniPrep, digested plasmids with RE Xho1 and Nde1. Expected size ~2600 bp. All? samples had postive inserts, sent sample 2 (lane 2) to sequencing
Date: July 17, 2012
Digested sample 2 again with Xho1 and Nde1 (see lane 3), excepted size 2600 bp. Too many bands?
Date: July 23, 2012
Prepared MiniPrep, digested plasmids with RE Xho1 and Nde1. Expected size ~2600 bp. Samples 1, 3, 4, 5 and 6 had postive inserts, sent sample 1 (lane 1) to sequencing
Result intepretation/Summary
[edit]
Conclusion
[edit]