Difference between revisions of "Team:Aachen/Notebook/Documentation/Glycogen Knockout Generation"
Lhelleckes (Talk | contribs) (→15-07-15) |
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==15-07-14== | ==15-07-14== | ||
− | {{Team:Aachen/DoubleFigure|Aachen_15-07-14_glgX_BL21_glgXP_BL21_knockout_edited_area.png|Aachen 15-07-14 glgX BL21 glgXP BL21 knockout genomic area.png|title1= |title2= |subtitle1= |subtitle2= |size= | + | {{Team:Aachen/DoubleFigure|Aachen_15-07-14_glgX_BL21_glgXP_BL21_knockout_edited_area.png|Aachen 15-07-14 glgX BL21 glgXP BL21 knockout genomic area.png|title1= |title2= |subtitle1= |subtitle2= |size=medium}} |
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* gel shows that all glgX double-knockouts in BL21 have worked | * gel shows that all glgX double-knockouts in BL21 have worked | ||
* just clone #1 of the single-knouckout in BL21 looks good | * just clone #1 of the single-knouckout in BL21 looks good | ||
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|- | |- | ||
|} | |} | ||
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+ | {{Team:Aachen/Figure|Aachen_15-07-18 glgXP knockout candidates colony PCR.png|title=PfuS PCR to check the double knockout candidates|subtitle=|size=medium}} | ||
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* overnight cultures with IPTG of good clones | * overnight cultures with IPTG of good clones | ||
* plate cured #3RZ1#, #ZPKP#, #PH3R# and #XHEK# on LB+K and LB+A (control) and make an LB{{sup|-}} overnight | * plate cured #3RZ1#, #ZPKP#, #PH3R# and #XHEK# on LB+K and LB+A (control) and make an LB{{sup|-}} overnight | ||
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* gel of PCR products: clones #2-4 look good and are purified (see table above) | * gel of PCR products: clones #2-4 look good and are purified (see table above) | ||
− | {{Team:Aachen/Figure|Aachen_15-07-20 glgXP PfuS genomic region.png|title=PfuS PCR of glgXP knockout candidates|subtitle=The genomic region of all clones was amplified with #6V6P# and #WAO8# for sequencing. Only clones 2-4 have the correct length. |size= | + | {{Team:Aachen/Figure|Aachen_15-07-20 glgXP PfuS genomic region.png|title=PfuS PCR of glgXP knockout candidates|subtitle=The genomic region of all clones was amplified with #6V6P# and #WAO8# for sequencing. Only clones 2-4 have the correct length. |size=medium}} |
* pipette for sequencing | * pipette for sequencing | ||
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* gel looks good | * gel looks good | ||
+ | {{Team:Aachen/Figure|Aachen_15-08-04 BL21 glgX knockout check, sequencing.png|title=BL21 Gold (DE3) Δ''glgX'' knockout check|subtitle=The genomic region of all clones was amplified with #6V6P# and #WAO8# for sequencing. Only clones 2-4 have the correct length. |size=medium}} | ||
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* PCR clean-up | * PCR clean-up | ||
* product in #LRCC# | * product in #LRCC# | ||
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products stored in #938K# | products stored in #938K# | ||
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+ | {{Team:Aachen/Figure|Aachen_15-08-12 glgXP BL21 PfuS PCR.png|title= PCR to check double knockout candidates |subtitle= |size=medium} | ||
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* no 1 and 2 looked good on the gel | * no 1 and 2 looked good on the gel | ||
** PCR purification (1: #A6XM#, #C3B3#) | ** PCR purification (1: #A6XM#, #C3B3#) | ||
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| final elongation || 68 || 5'00" | | final elongation || 68 || 5'00" | ||
|} | |} | ||
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+ | {{Team:Aachen/Figure|Aachen_15-08-13 glgXP knockout candidates BL21.png|title=BL21 Gold (DE3) Δ''glgX/P'' double knockouts check|subtitle= |size=medium}} | ||
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* run an agarose gel | * run an agarose gel | ||
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* PfuS PCR on knockout candidates (tube 7: water control) | * PfuS PCR on knockout candidates (tube 7: water control) | ||
+ | {{Team:Aachen/Figure|Aachen_15-08-12 15-08-14 bl21 glgxp double knockout pfuS PCR.png|title= PCR to check double knockout candidates |subtitle= |size=medium} | ||
* gel: tube 1,2,4 and 5 were good | * gel: tube 1,2,4 and 5 were good | ||
* PCR clean-up | * PCR clean-up |
Revision as of 19:29, 18 September 2015