LE392 and DH5alpha cultures in TSI Agar showed some growth. No black color change was visible, but both slants had a rotten egg scent, so suspected generation of H2S at concentrations too low to visibly trigger color change yet.
PCR work
Ran 1.0% agarose gel of four 6/30 PCR products at 90 V. Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
Based on gel, it appears that the phs50 thermocycler protocol is an improvement from phs protocol and that including DMSO also improves yield.
Miniprepped LE392 cultures grown overnight,resulting in two Eppendorfs apiece of the following plasmids: B0015 (double terminator), J23114 (constitutive promoter), pSB74 (thiosulfate reductase), R0011 (IPTG-inducible promoter), and P0312 (lacI needed with R0011).
Results of 6/30 Ligation (Ligation Attempt #1)
Plated post-ligation transformants showed the following results:
Reaction #1 (small control)
0 colonies
Reaction #2 (1:1 insert:vector)
2 colonies, numbered 1A and 2A
Reaction #3 (2:1 insert:vector)
0 colonies
Reaction #4 (large control)
2 colonies
Reaction #5 (large 1:1 insert vector)
2 colonies, numbered 3A and 4A
Concerned about the small number of colonies and low ratio of number of colonies from actual ligation to number of colonies from control ligation.
Started liquid cultures and index plates of four colonies from reactions 2 and 5.
Also set up colony PCR for all four colonies, spotting the template DNA into tubes with the following contents and running on thermocycler protocol phs50 [add to protocols page and link]
Component
Volume
Distilled Water
27 μL
Phusion 5x Buffer
10 μL
DMSO
1.5 μL
10 mM dNTPs
1 μL
10 uM phsABC_F primer
5 μL
10 uM phsABC_R primer
5 μL
Phusion Polymerase
0.5 μL
Template DNA
spotting
Total
50 μL
After PCR, ran 1.0% agarose gel of colony PCR reaction solutions at 90 V versus a 1 kb ladder. A preliminary visualization showed no visible non-primer DNA except for a band between 3 and 4 kb for the PCR product from colony 1. Based on a later visualization said band appeared to be closer to 3 kb, suggesting the 3.2 kb B0015 vector rather than the 3.6 kb insert. Unfortunately gel was dropped and damaged irreparably before picture could actually be taken.
Wetlab work for this day is also described on pages 37-41 of the hard copy lab notebook
Project name
Main project page
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