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

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


Improved Fsp Digest

Fsp digest, zoomed in
Fsp digest, zoomed in
Fsp digest,
Fsp digest,
Fsp digest, brighter
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, Yellow group
T/R Gel, Orange group
T/R Gel, Orange group
T/R Gel, Blue group
T/R Gel, Blue group
T/R Gel, Grey group
T/R Gel, Grey group
T/R Gel, Red group
T/R Gel, Red group
T/R Gel, Pink group
T/R Gel, Pink group
Teaching example
Teaching example
T/R Gel, Green group
T/R Gel, Green group
T/R Gel, Purple group
T/R Gel, Purple group


Teaching example, cropped
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, Yellow group
W/F Gel, Orange group
W/F Gel, Orange group
W/F Gel, Blue group
W/F Gel, Blue group
W/F Gel, Grey group
W/F Gel, Grey group
W/F Gel, Red group
W/F Gel, Red group
W/F Gel, Red group
W/F Gel, Red group
W/F Gel, Pink group
W/F Gel, Pink group
W/F Gel, Green group
W/F Gel, Green group
W/F Gel, Purple group
W/F Gel, Purple group
W/F Gel, Red group done at 50 °C
W/F Gel, Red group done at 50 °C


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

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