1206 pET28b-recT
Cloning of 1206 pET28b-recT
[edit]Purpose: Cloning of 1206 pET28b-recT
Date: June 26, 2012
Notebook:
Page:
Clone ID: 1206
Experimenters: Jennifer Truong
Results
[edit]Cloning
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Amplified DNA with PHUSION enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature)
Cut out band in lane 3 (expected size = 834 bp), purified and digested wth vector 12 (pET28b) with restriction enzymes Xho1 and Nhe1
Cut out insert from lane 3 and vector from lane 4, purified and ligated overnight. Transform with Top10 cells and plate on KAN plates tomorrow
Date: July 18, 2012
Cut out band in lane 1 and 2 (expected size = 834 bp), purified and digested wth vector 12 (pET28b) with restriction enzymes Xho1 and Nhe1
Cut out insert from lane 1 and vector from lane 2, purified and ligated overnight. Transform with Top10 cells and plate on KAN plates tomorrow
Colony Screening
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Screened colonies, all positve, expected ize ~800 bp
Grew lanes/sample 1-6 in media in shaker overnight, MiniPrep tomorrow.
Date: June 29, 2012
Digested MiniPrep with Nhe1 + Xho1, sent sample 3 (lane 9) to sequencing
looks like the digestion is not complete (two big bands), need to digest a little bit longer or add more enzyme
Date: July 12, 2012
Sequencing came back bad, grow more bacteria from stock solution, redid MiniPrep, digested plasmids with RE Xho1 and Nhe1 (see lanes 3 and 4). Expected size ~800 bp. Both samples had postive inserts, sent sample 2 (lane 4) to sequencing
Date: July 16, 2012
Sequencing came back bad, grow more bacteria from plate, redid MiniPrep, digested plasmids with RE Xho1 and Nhe1. Expected size ~800 bp. All samples had postive inserts?
Date: July 17, 2012
Digested sample 4 with Xho1 and Nhe1 (see lane 4), expected size ~800 bp. Too many bands
Date: July 20, 2012
Screened colonies for positive inserts
Grew samples 1, 2, 3, 5, 6 and 7 in media and put in shaker overnight
Date: July 23, 2012
Prepared MiniPrep, digested plasmids with RE Xho1 and Nhe1. Expected size ~800 bp. All samples had postive inserts, sumbitted sample 6 (lane 6) for sequencing
Result intepretation/Summary
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Conclusion
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