Difference between revisions of "Team:Aachen/Notebook/Documentation/Glycogen Knockout Generation"
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** glgX #OM6C# with #6V6P# and #WAO8# 55-64 °C (expected: 1024, maybe: 1006) > products in #LA33#, 63.5 is the best annealing temperature | ** glgX #OM6C# with #6V6P# and #WAO8# 55-64 °C (expected: 1024, maybe: 1006) > products in #LA33#, 63.5 is the best annealing temperature | ||
** glgP #VZD3# with #T9T9# and #CT4E# 55-64 °C (expected: 1015, maybe: 3351) > products in #DOMW# | ** glgP #VZD3# with #T9T9# and #CT4E# 55-64 °C (expected: 1015, maybe: 3351) > products in #DOMW# | ||
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+ | {{Team:Aachen/DoubleFigure|Aachen_15-06-16_genomic_gradient_glgX.png|Aachen_15-06-16_glgP_colony PCR.png|title1=Gradient PCR on genomic region of ''glgX''|title2=Gradient PCR on genomic region of ''glgP''|subtitle1=PCR worked at all temperatures.|subtitle2= PCR worked at all temperatures.|size=medium}} | ||
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'''30 cycles''' | '''30 cycles''' | ||
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==15-06-17== | ==15-06-17== | ||
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| template DNA || 2 µl per reaction || | | template DNA || 2 µl per reaction || | ||
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* PCR product purification | * PCR product purification | ||
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+ | {{Team:Aachen/Figure|Aachen_15-07-01_BL21_glgP_colony_PCR.png|title=Colony PCR on ''glgP'' knockout candidates|subtitle=The Colony PCR was not very succesful. Nevertheless, three candidates (4, 18, 20) look good.|size=large}} | ||
The colony PCR was not very successful. Many samples did not work, including the controls. Nevertheless, we have identified three candidates (4, 18, 20). | The colony PCR was not very successful. Many samples did not work, including the controls. Nevertheless, we have identified three candidates (4, 18, 20). | ||
Overnight cultures of good knockout candidates 4, 18, 20. 2 ml of LB+K+IPTG were inoculated with 2 µl of suspended cells | Overnight cultures of good knockout candidates 4, 18, 20. 2 ml of LB+K+IPTG were inoculated with 2 µl of suspended cells | ||
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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=glgXP double knockout and glgX single knockout in BL21|title2= glgXP double knockout and glgX single knockout in BL21|subtitle1=The primer SNYO binds specifically on the iGEM Aachen MultiSTOP and therefore indicates the insertion of this fragment. All double knockout candidates look good, but only #1 of the double knockout candidates has the correct length. |subtitle2=The primer 6V6P binds in the genomic area, so this is a control beacuse a band is present whether the gene is disrupted or not. |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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* PCR product purification | * PCR product purification | ||
+ | {{Team:Aachen/Figure|Aachen_15-07-16 PfuS pcr XP double and X single knockout in BL21.png|title=PfuS PCR to check the ''glgX'' and the double knockout candidates|subtitle=|size=medium}} | ||
{|class="wikitable" | {|class="wikitable" | ||
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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_15-08-14 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 | ||
* pipette for sequencing | * pipette for sequencing | ||
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=Results= | =Results= |
Latest revision as of 20:27, 18 September 2015