1148 pTXB1-CH
Cloning of 1148 pTXB1-CH
[edit]Purpose: Cloning of 1148 pTXB1-CH
Date: June 26, 2012
Notebook:
Page:
Clone ID: 1148
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 2 (expected size = 784 bp), purified and digested wth vector 1126 (pTXB1) with restriction enzymes Xho1 and Pst1
I don't know what happened to the digested insert (lane 1), will redo tomorrow. Cut out digested vector (lane 2) to save
Date: June 27, 2012
Reamplified DNA with PHUSION enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature)
Cut out band in lane 3 (expected size = 784 bp), purified and digested wth vector 1126 (pTXB1) with restriction enzymes Xho1 and Pst1
Digested 1148 DNA and pTXB1 vector with restriction enzymes Xho1 and Pst1
See lanes 2 and 3. Cut out insert from lane 2, and cut out top vector band in lane 3. Purified and ligated together. Transformed with Top10 cells, plated on Amp resistant plates, grow overnight.
Date: July 18, 2012
Started all over becuase this experiment was started before the power went out. Samples may have problems
Cut out band in lanes 1 and 2 (expected size = 784 bp), purified and digested wth vector 1126 (pTXB1) with restriction enzymes Xho1 and Pst1
See lanes 1 and 2. Cut out insert from lane 1, and cut out top vector band in lane 2. Purified and ligated together. Transformed with Top10 cells, plated on Amp resistant plates, grow overnight.
Colony Screening
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Screened colonies.
Samples 1, 2, 3, 4, 6 and 7 were positive (same respected lanes), expected size ~800 bp
Grew in media in shaker overnight.
Screening was bad, redo
Date: July 12, 2012
Sequencing came back bad, grow more bacteria from stock solution, redid MiniPrep, digested plasmids with RE Xho1 and Pst1 (see lanes 1 and 2). Expected size ~800 bp. Both samples had postive inserts, sent sample 1 to sequencing
Date: July 16, 2012
Sequencing came back bad, grow more bacteria from plate, redid MiniPrep, digested plasmids with RE Xho1 and Pst1. Expected size ~800 bp. All samples had postive inserts?
July 17, 2012
Digested sample 4, see lane 5 again with Xho1 and Pst, expected size ~800 bp. No insert
Date: July 19, 2012
Samples 1, 2, 3, 4, 5 and 6 were positive (same respected lanes), expected size ~800 bp
Grew in media in shaker overnight.
Date : July 20, 2012
Digested MiniPrep plasmids with RE Xho1 and Pst1. Expected size ~800 bp. All samples had postive inserts? Sent sample 1 to sequencing
Result intepretation/Summary
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Conclusion
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