Talk:20.109(S09):Induce protein and evaluate DNA (Day5)

Examples of induced (left) and control (right) cell pellets

Improved Fsp Digest

Fsp digest, zoomed in

Fsp digest,

Fsp digest, brighter

Lane 1 is the 1 Kbp ladder, lane 5 the 100 bp ladder.

Lanes 2, 3, and 4 are uncut IPC (WT), digested IPC, and digested IPC-S101L, respectively.

Samples were digested overnight, as FspI appears to digest somewhat weakly otherwise.

TR O/N cultures and Gels

The table below lists the OD₆₀₀ values of 1:10 dilutions of the cultures grown overnight, as well as the pellet observations.

Group	OD WT	OD S101L	OD Chosen mutant	Pellets yellow?
Pink	0.117	0.098	0.126	Yes
Yellow	0.118	0.091	0.148	WT and S101L bright yellow-green; Y64D paler matte yellow
Red	0.121	0.187	0.129	Yes
Orange	0.117	0.087	0.122	WT and S101L bright yellow-green; E31I paler matte yellow
Purple	0.109	0.075	0.121	Yes
Green	0.106	0.095	0.072	Yes
Grey	0.111	0.102	0.148	All bright yellow-green

Note: 1% gels were run for 45 min, 1.2% gels for 60 min, both at 100V.

T/R Gel, Yellow group

T/R Gel, Orange group

T/R Gel, Blue group

T/R Gel, Grey group

T/R Gel, Red group

T/R Gel, Pink group

Teaching example

T/R Gel, Green group

T/R Gel, Purple group

Teaching example, cropped

There seems to have been a contamination or other strange issue with the S101L DNA and/or FspI enzyme. For your final reports, please use my old data, which is posted above. Unfortunately, some of the bands are faint (this experiment pre-dates our modern gel doc system). Lane 1 is parent IPC (only one form of the plasmid is visible), lane 2 is the ladder, lane 4 is the parent digested with FspI, and lane 8 is the S101L mutant digested with FspI. The short band is very faint, but it is present. I will have the original photo available for viewing for anyone who wants to clearly see the short band. At right is a cropped version for easier viewing. Update: Upon reflection, most of the S101L data does not look nearly as bad as I thought. As you interpret your data, keep in mind what are all the possible band sizes you could get. What if the enzyme only partially digests? Nevertheless, I will try to re-run this experiment to get a better photo for your reports in the coming days.

WF O/N cultures and Gels

The table below lists the OD₆₀₀ values of 1:10 dilutions of the cultures grown overnight, as well as the pellet observations.

Group	OD WT	OD S101L	OD Chosen mutant	Pellets yellow?
Pink	0.105	0.098	0.105	Yes
Purple	0.244	0.121	0.122	Yes
Red (O/N)	0.229	0.284	0.282	WT and S101L bright yellow-green, mutant white
Grey (O/N)	0.206	0.106	0.109	All are bright yellow-green
Green	0.245	0.099	0.115	all are yellow/green
Yellow	0.238	0.120	0.116	all are bright yellow-green

W/F Gel, Yellow group

W/F Gel, Orange group

W/F Gel, Blue group

W/F Gel, Grey group

W/F Gel, Red group

W/F Gel, Red group

W/F Gel, Pink group

W/F Gel, Green group

W/F Gel, Purple group

W/F Gel, Red group done at 50 °C

Please see notes about S101L in T/R section above.