Paul.xu at 16:42, 19 June 2020

2020-06-19T16:42:36Z

New page

[[File:2746 2770 2779 PCR.JPG|RTENOTITLE]]

[[File:2746 2662 2783 NdeI XhoI.JPG|RTENOTITLE]]

[[File:2746 NcoI XhoI.JPG|RTENOTITLE]]

20200514 2746 pminiT2 NEBturbo miniprep EcoRI

[[File:2746 miniprep ECoRI.JPG|RTENOTITLE]]2746 PCR

[[File:2746 PCR 2743 2743 Gigaprep.JPG|RTENOTITLE]]

[[File:2723 2742 2752 2746 digestion.JPG|RTENOTITLE]]

[[File:2746 subcloning.JPG|RTENOTITLE]]

pET28-3XFlag-N miniprep digestion

[[File:2746 digestion2.JPG|RTENOTITLE]][[File:2746 digestion1.JPG|RTENOTITLE]]

PCR, 6/5/2020

[[File:2778 2746 PCR.JPG|RTENOTITLE]]

[[File:2746 2760 EcoRI.JPG|RTENOTITLE]]

[[File:2746 pMiniT2 NdeI XhoI.JPG|RTENOTITLE]]

[[File:2772 2746 2779 2778.JPG|RTENOTITLE]]

[[File:2746 2795 gibson.JPG|RTENOTITLE]] [[File:2746 gibson vector.JPG|RTENOTITLE]]

ignore this 2696-pet28b vector. it failed

[[File:2746 gibson.JPG|RTENOTITLE]]

[[File:2746 pET28b NcoI XhoI.JPG|RTENOTITLE]]

== 2746-pJet1.2 and subclone ==

[[File:2746 PCR.JPG|RTENOTITLE]]

20200616 2746 pJet NcoI-HF+XhoI in Cutsmart, 37oC, 1hour

[[File:2746 2763 2773 2779 pJet digestion R.JPG|RTENOTITLE]]

[[File:2746 2696 NcoI XhoI.JPG|RTENOTITLE]]

20200617 2746-pJet2.1 digested with NcoI+XhoI. But two bands, one is between 1-2kb and the other one around 3kb, can be seen and cut for ligation with vector pET28b- seperately. Which one is the correct insert will be further determined.

[[File:2746 2773 2779 2795 pJet digestion.JPG|RTENOTITLE]]

20200617 2696/940/2696 all three vectors can provide pET28b-. Select 2696 for further subclone.
[[File:2746 Subclone NcoI XhoI.JPG|RTENOTITLE]]

20200619 2746-pET28b clone PCR was performed. Primers used will produce 1385bp product. So the insert band between 1-2kb looks like the one we want. 

[[File:2746 2795 clone PCR.JPG|RTENOTITLE]]
[[Category:Cloning]]

2746 - Revision history

Paul.xu at 16:42, 19 June 2020