SBB12Ntbk-SpencerScott: Difference between revisions
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# Sequencing was perfect for all reporter constructs | # Sequencing was perfect for all reporter constructs | ||
# Started Bsrs047 and Bsrs048 comp cell cultures in 2YT-Spec at 8:48am | # Started Bsrs047 and Bsrs048 comp cell cultures in 2YT-Spec at 8:48am | ||
# Did 6 total Expand EIPCR's with the '55' thermo program on 9145-Bsrs038, 9525-Bsrs038, & 9525-Bsrs044 (2 on each) (should be done around 11:15am)<br> [[ | # Did 6 total Expand EIPCR's with the '55' thermo program on 9145-Bsrs038, 9525-Bsrs038, & 9525-Bsrs044 (2 on each) (should be done around 11:15am)<br> [[Image:20120124 13809(small).png|100px]] | ||
# Digested; Picked lanes 1, 3, 5, 7 & 8 | # Digested; Picked lanes 1, 3, 5, 7 & 8 | ||
# Purified, Ligated 1 with 8 and 5 with 7 | # Purified, Ligated 1 with 8 and 5 with 7 | ||
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# Great sequencing! Got all of our parts | # Great sequencing! Got all of our parts | ||
# Bam/xhoi& BgIII/xhoi digested 1600-Bsrs045/Bsrs046& 1600-jtk2541:<br> [[ | # Bam/xhoi& BgIII/xhoi digested 1600-Bsrs045/Bsrs046& 1600-jtk2541:<br> [[Image:20120224 13610(small).png|100px]] | ||
# (1486, 1227, S) & (2239, 1149, L) | # (1486, 1227, S) & (2239, 1149, L) | ||
# Ligated; Transformed into MC1061; Recovered; Plated on Spec | # Ligated; Transformed into MC1061; Recovered; Plated on Spec | ||
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# 24 mini-preps! | # 24 mini-preps! | ||
# 24 Eco/Bam Digestions! | # 24 Eco/Bam Digestions! | ||
# Lengths: '''1-4:''' (2635, 753); '''5-10:''' (2066, 615); '''11-16:''' (2481, 615); '''17-24:''' (2481, 1108)<br> [[ | # Lengths: '''1-4:''' (2635, 753); '''5-10:''' (2066, 615); '''11-16:''' (2481, 615); '''17-24:''' (2481, 1108)<br> [[Image:20120223 13600(small).png|100px]][[Image:20120223 13601(small).png|114px]] | ||
# They all worked except the last one. Sent in for sequencing 1, 2, 8, 9, 10, 11, 12, 14, 17, 19, 21, & 22. | # They all worked except the last one. Sent in for sequencing 1, 2, 8, 9, 10, 11, 12, 14, 17, 19, 21, & 22. | ||
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# The PCR's of LexA look good. I will try to re-digest and re-ligate them. | # The PCR's of LexA look good. I will try to re-digest and re-ligate them. | ||
# Need to move JTK2541 into 1600 and 1601CA so I can ligate it with SulA promoters. | # Need to move JTK2541 into 1600 and 1601CA so I can ligate it with SulA promoters. | ||
# Eco/Bam, JTK2541, 1600, 1601CA, 9145, 9525, & LexA (w.t)<br> (1) (2481+753, S)<br> (2) (2635+78, L)<br> (3) (3205+78, L)<br> (4) (2066+342, L)<br> (5) (2481+1854, L) <br> (6) (615)<br> [[ | # Eco/Bam, JTK2541, 1600, 1601CA, 9145, 9525, & LexA (w.t)<br> (1) (2481+753, S)<br> (2) (2635+78, L)<br> (3) (3205+78, L)<br> (4) (2066+342, L)<br> (5) (2481+1854, L) <br> (6) (615)<br> [[Image:20120221 13566(small).png|100px]] | ||
# Eco/Nhe1 LexA(1-87)'s and 9525-1834<br> (1) 261 <br> (2) 261 <br> (3) (3313+1022, L)<br> [[ | # Eco/Nhe1 LexA(1-87)'s and 9525-1834<br> (1) 261 <br> (2) 261 <br> (3) (3313+1022, L)<br> [[Image:20120221 13565(small).png|100px]] | ||
#Gel Purify | #Gel Purify | ||
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# Picked 4 colonies for each SulA promoter grew in CA for the 1601CA and Spec for the 1600.<br> Also picked four non-red 9145-Bsrs038 cells, grew in AMP. | # Picked 4 colonies for each SulA promoter grew in CA for the 1601CA and Spec for the 1600.<br> Also picked four non-red 9145-Bsrs038 cells, grew in AMP. | ||
# Set up the titrations | # Set up the titrations | ||
# Eco/Bam'd the 8 Lex(1-87) to map. (desired size: 2400,1100)<br> [[ | # Eco/Bam'd the 8 Lex(1-87) to map. (desired size: 2400,1100)<br> [[Image:20120218 13538(small).png|100px]]<br> Lanes 2, 3, 5, & 8 looked promising; I realized that they shouldn't be red because there is not promoter, so anything that was red like lane 1 is most likely background; i sent for sequencing since I want to have this part anyway. | ||
# Sent for sequencing 1, 2, 3, 5, 8, Bss003, Bss006, and the mixed plasmids reading from ca56 | # Sent for sequencing 1, 2, 3, 5, 8, Bss003, Bss006, and the mixed plasmids reading from ca56 | ||
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# Eco/Bam'd the two Wobble reactions and 1601CA-Bss52.<br> I accidentally put 7 ul of Part for 1601CA instead of 3 so I added .5ul of NEB2 in addition to the original .5, such that it should be around 10% of the volume. | # Eco/Bam'd the two Wobble reactions and 1601CA-Bss52.<br> I accidentally put 7 ul of Part for 1601CA instead of 3 so I added .5ul of NEB2 in addition to the original .5, such that it should be around 10% of the volume. | ||
# Sequencing data came back; I forgot to specify primers so the only read I got was ca56 off Bss006 which looked great. | # Sequencing data came back; I forgot to specify primers so the only read I got was ca56 off Bss006 which looked great. | ||
# Gel purified (small frag purified for the sula promoters)<br> [[ | # Gel purified (small frag purified for the sula promoters)<br> [[Image:20120217 13504(small).png|100px]] | ||
# The 1601CA was kinda weak so I also used a E/Bam digest of 1600 from Gabe that might also be sketchy. I ligated each of the sulA promoters into these plasmids. (4 ligations) | # The 1601CA was kinda weak so I also used a E/Bam digest of 1600 from Gabe that might also be sketchy. I ligated each of the sulA promoters into these plasmids. (4 ligations) | ||
# Transformed into MC1061; Recovered for 30 mins in 2YT | # Transformed into MC1061; Recovered for 30 mins in 2YT | ||
# Plated 1601CA on CA and 1600 on Spec. | # Plated 1601CA on CA and 1600 on Spec. | ||
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Revision as of 10:32, 19 March 2012
SpencerScott 10:29, 19 March 2012 (PDT)
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SpencerScott 11:59, 15 March 2012 (PDT)
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SpencerScott 13:50, 11 March 2012 (PDT)
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SpencerScott 10:07, 12 March 2012 (PDT)
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SpencerScott 13:50, 11 March 2012 (PDT)
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SpencerScott 11:41, 10 March 2012 (PST)
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SpencerScott 13:51, 9 March 2012 (PST)
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SpencerScott 15:00, 6 March 2012 (PST)
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SpencerScott 15:00, 6 March 2012 (PST)
Spencer Is in New Zealand 2/24-3/6
SpencerScott 14:59, 6 March 2012 (PST)
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SpencerScott 10:18, 23 February 2012 (PST)
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SpencerScott 20:37, 22 February 2012 (PST)
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SpencerScott 15:19, 20 February 2012 (PST)
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SpencerScott 12:33, 18 February 2012 (PST)
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SpencerScott 12:33, 17 February 2012 (PST)
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