Editing 1206 pET28b-recT

== '''Cloning of 1206 pET28b-recT'''  ==

'''Purpose:''' Cloning of 1206 pET28b-recT 

'''Date: '''June 26, 2012<br> 

'''Notebook:&nbsp;''' 

'''Page:''' 

'''Clone ID: 1206''' 

'''Experimenters: '''Jennifer Truong 

== '''Results'''  ==

=== Cloning  ===

[[Image:0626 Amp.JPG]]<br> 

Amplified DNA&nbsp;with PHUSION&nbsp;enzyme (0.5 uL , 25 cycles, 55 degees annealing temperature)<br> 

Cut out band in lane 3 (expected size = 834 bp), purified and digested wth vector 12 (pET28b)&nbsp;with restriction enzymes Xho1 and Nhe1<br> 

[[Image:0626 Digestion.JPG]]<br> 

Cut out insert from lane 3 and vector from lane 4, purified and ligated overnight. Transform with Top10 cells and plate on&nbsp;KAN&nbsp;plates tomorrow 

<br> 

Date:&nbsp;July 18, 2012<br> 

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<br> 

[[Image:1209 0718 Amp.JPG]] 

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 <br> 

[[Image:1206 0718 Digestion.JPG]] 

=== <u></u>Colony Screening  ===

[[Image:1206 Screening.JPG]]<br> 

Screened colonies, all positve, expected ize ~800 bp 

Grew lanes/sample 1-6 in media in shaker overnight, MiniPrep tomorrow. 

Date:&nbsp;June 29, 2012 

[[Image:0629 Screening.JPG]] 

Digested MiniPrep with Nhe1 + Xho1, sent sample 3 (lane 9)&nbsp;to sequencing 

'''looks like the digestion is not&nbsp; complete (two big bands), need to digest a little bit longer or add more enzyme''' 

Date:&nbsp;July 12, 2012<br> 

[[Image:0712 Screening.JPG]]<br> 

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<br> 

Date:&nbsp;July 16, 2012<br> 

[[Image:1206 0716 Screening.JPG]]<br> 

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?<br> 

Date:&nbsp;July 17, 2012 

[[Image:0717 Digestion.JPG]] 

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 

[[Image:1206 0720 Screening.JPG]] 

Grew samples 1, 2, 3, 5, 6 and 7 in media and put in shaker overnight 

Date:&nbsp;July 23, 2012<br>

[[Image:1206_0623_Screening.JPG]]<br>

Prepared MiniPrep, digested plasmids with RE Xho1 and Nhe1. Expected size ~800 bp. All samples had postive inserts, sumbitted sample 6 (lane 6)&nbsp;for sequencing<br>

== '''Result intepretation/Summary'''  ==

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== '''Conclusion'''  ==

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[[Category:Cloning]]