In order to determine success of ligation attempts 1 and 2, digested the miniprepped plasmids 1A-4A and 1B-9B from 7/5 with XbaI and SpeI and then looked for evidence of two fragments, one at 3.2 kb and the other at 3.6 kb. Reactions were conducted for 2 hours at 37°C, with the following contents for each of the thirteen reactions
Component
Volume
Distilled Water
6.4 μL
10x NEB Buffer 4
2 μL
DNA solution
10 μL
NEB XbaI
0.8 μL
NEB SpeI
0.8 μL
Total
20 μL
The diagnostic 1.0% agarose gel was run at 90 V vs. a 1 kb ladder. Each of the thirteen samples gave one diffuse band around 3 kb and no evidence of the 3.6 kb phsABC fragment, so both ligation attempts #1 and #2 were unsuccessful. Gel of XbaI and SpeI double digest of plasmids from ligation attempts #1 and #2 Lane 1: 1 kb ladder, Lanes 2-5:1A-4A, Lanes 6-14: 1B-9B
During digestion prepped plasmid samples for sequencing. Each sample contained 3 μL of plasmid soln (average concentration of 96 ng/μL), 2 μL of 4 mM VF2 (forward primer), and 13 μL of distilled water. Intended to follow up with reverse sequencing of more promising samples, but gel results made it a moot point.
Wetlab work for this day is also recorded on pages 47, 49, and 50 in the hard copy lab notebook.
Project name
Main project page
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