Talk:20.109(S09):Induce protein and evaluate DNA (Day5): Difference between revisions
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[[Image:20.109 induced-pellets.jpg|thumb|500px|center|Examples of induced (left) and control (right) cell pellets]] | [[Image:20.109 induced-pellets.jpg|thumb|500px|center|Examples of induced (left) and control (right) cell pellets]] | ||
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==Improved Fsp Digest== | |||
[[Image:S09_FspDigest-zoom.BMP | right |thumb|250px | Fsp digest, zoomed in ]] | |||
[[Image:S09_FspDigest.BMP | left |thumb|250px | Fsp digest, ]] | |||
[[Image:S09_FspDigest-bright.BMP | center |thumb|250px | Fsp digest, brighter]] | |||
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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. | |||
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! Group | ! Group | ||
! OD WT | ! OD WT | ||
! OD | ! OD S101L | ||
! OD | ! OD Chosen mutant | ||
! Pellets yellow? | ! Pellets yellow? | ||
|- | |- | ||
|Pink | |Pink | ||
|0.117 | |0.117 | ||
|0.098 | |||
|0.126 | |0.126 | ||
|Yes | |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 | |||
|} | |} | ||
</center> | </center> | ||
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[[Image:S09_M1D3_TR_Red.BMP | left |thumb|250px | T/R Gel, Red group]] | [[Image:S09_M1D3_TR_Red.BMP | left |thumb|250px | T/R Gel, Red group]] | ||
[[Image:S09_M1D3_TR_Pink.BMP | center |thumb|250px | T/R Gel, Pink group]] | [[Image:S09_M1D3_TR_Pink.BMP | center |thumb|250px | T/R Gel, Pink group]] | ||
[[S09-M1D5 teaching-ex.BMP |right |thumb|250px | Teaching example]] | [[Image:S09-M1D5 teaching-ex.BMP |right |thumb|250px | Teaching example]] | ||
[[Image:S09_M1D3_TR_Green.BMP | left |thumb|250px | T/R Gel, Green group]] | [[Image:S09_M1D3_TR_Green.BMP | left |thumb|250px | T/R Gel, Green group]] | ||
[[Image:S09_M1D3_TR_Purple.BMP | center |thumb|250px | T/R Gel, Purple group]] | [[Image:S09_M1D3_TR_Purple.BMP | center |thumb|250px | T/R Gel, Purple group]] | ||
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. 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. | <br style="clear:both;"/> | ||
[[Image:S09-M1D5_S101L-cropped.tiff|right |thumb|100px | 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. <font color=990000>'''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.'''</font color> | |||
<br style="clear:both;"/> | <br style="clear:both;"/> | ||
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! Group | ! Group | ||
! OD WT | ! OD WT | ||
! OD | ! OD S101L | ||
! OD | ! OD Chosen mutant | ||
! Pellets yellow? | ! 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 | |||
|} | |} | ||
</center> | </center> | ||
<br style="clear:both;"/> | <br style="clear:both;"/> | ||
[[Image:S09_M1D5_WF_Yellow.BMP | right |thumb|250px | W/F Gel, Yellow group]] | |||
[[Image:S09_M1D5_WF_Orange.BMP | left |thumb|250px | W/F Gel, Orange group]] | |||
[[Image:S09_M1D5_WF_Blue.BMP | center |thumb|250px | W/F Gel, Blue group]] | |||
[[Image:S09_M1D5_WF_Grey.BMP | right |thumb|250px | W/F Gel, Grey group]] | |||
[[Image:S09_M1D5_WF_Red1.BMP | left |thumb|250px | W/F Gel, Red group]] | |||
[[Image:S09_M1D5_WF_Red2.BMP | center |thumb|250px | W/F Gel, Red group]] | |||
[[Image:S09_M1D5_WF_Pink.BMP | right |thumb|250px | W/F Gel, Pink group]] | |||
[[Image:S09_M1D5_WF_Green.BMP | left |thumb|250px | W/F Gel, Green group]] | |||
[[Image:S09_M1D5_WF_Purple.BMP | center |thumb|250px | W/F Gel, Purple group]] | |||
[[Image:S09_M1D5_WF_Red-redo.BMP | center |thumb|250px | W/F Gel, Red group done at 50 °C]] | |||
<br style="clear:both;"/> | |||
Please see notes about S101L in T/R section above. |
Latest revision as of 20:07, 9 March 2009
Improved Fsp Digest
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.
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 |
Please see notes about S101L in T/R section above.