1148 pTXB1-CH

Cloning of 1148 pTXB1-CH

Purpose: Cloning of 1148 pTXB1-CH

Date: June 26, 2012

Notebook: 

Page:

Clone ID: 1148

Experimenters: Jennifer Truong

Results

Cloning

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.

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Colony Screening

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