2012_11_29 Second 13C formate --> protein experiment
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2012_11_ 30 10am: do the t= 15 hours of induction gel. This will see if induction is observed, and see if we no longer need to pre-run gels if we make them with the commercial BioRad Tris buffers when pouring gelsSample:Strainhrs after some were inducedinduced?time protein alliquot was collecteduL culture collecteduL of 4x buffer + beta-ME addedtotal volumeHow much beta-ME do I add? Want 1%, so use (volume of buffer)*0.01*4 and it will be 1% in 1X
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INDUCED ONLY, 15 hours:
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Pseudo Strain: Amanda is thawing some pellets to get more biomass for the pseudo strain. We plan to make a bunch so we can have one batch that will last a while.8-13 control: induced 15 hours: 0.5mM IPTG3 control15yes8:5012040160
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How many uL of sample should I load? Look at the last gel (11/27). 7 uL of a culture that had been induced for 24 hours was not overloaded. Since these have only been going 15 hours, let's do 8.5 uL.8-23: induced 15 hours: 0.5mM IPTG315yes8:5012040160
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8-35 control: induced 15 hours: 0.5mM IPTG5 control15yes8:5012040160
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8-45: induced 15 hours: 0.5mM IPTG515yes8:5012040160
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WellSampleDescriptionuL8-53 control: induced 15 hours: 1mM IPTG3 control15yes8:5012040160
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1ladder8-63: induced 15 hours: 1mM IPTG315yes8:5012040160
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28-13 control: induced 15 hours: 0.5mM IPTG8.58-75 control: induced 15 hours:1mM IPTG5 control15yes8:5012040160
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38-23: induced 15 hours: 0.5mM IPTG8.58-85: induced 15 hours: 1mM IPTG515yes8:5012040160
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48-35 control: induced 15 hours: 0.5mM IPTG8.5Use a non-black marker to write on tubeAmt required:320uL
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58-45: induced 15 hours: 0.5mM IPTG8.50.5 mM IPTG:beta-ME:12.8uL
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6new pseudo strain! prep 11/307.58-13 control: uninduced, 24 hours3 control~ 24NO
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78-53 control: induced 15 hours: 1mM IPTG8.58-23: uninduced, 24 hours3~ 24NO
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88-63: induced 15 hours: 1mM IPTG8.58-35 control: uninduced, 24 hours5 control~ 24NO
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98-75 control: induced 15 hours:1mM IPTG8.58-45: uninduced, 24 hours5~ 24NO
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108-85: induced 15 hours: 1mM IPTG8.51 mM IPTG:
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8-53 control: uninduced, 24 hours3 control~ 24NO
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8-63: uninduced, 24 hours3~ 24NO
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3pm: check the induced cells: use more of today's cultures & less pseudo strain8-75 control: uninduced, 24 hours5 control~ 24NO
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Well8-85: uninduced, 24 hours5~ 24NO
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1ladder5Use a non-black marker to write on tube
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28-13 control: induced, 24 hours, 0.5mM IPTG100.5 mM IPTG:
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38-23: induced, 24 hours, 0.5mM IPTG108-13 control: induced, 24 hours, 0.5mM IPTG3 control~ 24yes
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48-35 control: induced, 24 hours, 0.5mM IPTG108-23: induced, 24 hours, 0.5mM IPTG3~ 24yes
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58-45: induced, 24 hours, 0.5mM IPTG108-35 control: induced, 24 hours, 0.5mM IPTG5 control~ 24yes
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6new pseudo strain! prep 11/3068-45: induced, 24 hours, 0.5mM IPTG5~ 24yes
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78-53 control: induced, 24 hours, 1mM IPTG101 mM IPTG:
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88-63: induced, 24 hours, 1mM IPTG108-53 control: induced, 24 hours, 1mM IPTG3 control~ 24yes
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98-75 control: induced, 1mM IPTG108-63: induced, 24 hours, 1mM IPTG3~ 24yes
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108-85: induced, 24 hours, 1mM IPTG108-75 control: induced, 1mM IPTG5 control~ 24yes
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8-85: induced, 24 hours, 1mM IPTG5~ 24yes
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J strain:Description:
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39"strain 3": BL21(DE3) + pJ52 + Amanda's "3K3 J23100 cmFDH J23114 FocA #4"
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40"strain 5": BL21(DE3) + pJ52 + Amanda's "3K3 J23114 cmFDH J23114 FocA #4"
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41"strain 3 control": pSB1A3 EV & Amanda's "3K3 J23100 cmFDH J23114 FocA #4"
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42"strain 5 control": pSB1A3 EV & Amanda's "3K3 J23114 cmFDH J23114 FocA #4"
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Prep crude extract pseudo strain:
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1/2 mL of culture of broken cells (not crude extract so it will be less viscous.) Based on her other gel, in which 5 uL of 1/2 as concentrated sample looked good: mix 250 uL sample (yellow tube), 500 uL of water, and (750uL)*(1/3) of buffer with beta-ME. Load 7 - 8 uL/well.
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Mix enough BioRad buffer for this and the other 3 strip tubes:
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Sample uL mix needed
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pseudo strain250
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8*120 uL * 3strips960
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sum1210
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amt to prep:1331uL
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amt of Beta-ME to add:13.31uL
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Protein Gels 11-30
11/30 bioscreen plan
12/4/12 Actual Bioscreen layout
strains.csv
media.csv
wells.csv
Sample collection OD normalize
12/4 transfer to 13C media
12/1 growth to mid log
Protein Gels 12-2
copy: strains sheet
12/6: strains to grow
12/11 repeat 12/6-12/10
derivitization
Medias