SBB12Ntbk-SpencerScott: Difference between revisions
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==~~!~~== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Friday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Re-seed in morning for large scale comp cells. | |||
# make large scale comp cells! | |||
# Re-do all the transformations! '''Remember controls!!!''' | |||
| | |||
# | |||
|} | |||
==~~!~~== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Thursday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Pick in the morning. '''Remember controls!!''' | |||
# Decided to forgo making large scale comp cells from things that I haven't sequenced. I will make comp cells from my transformation today of my sequenced Bsrs051 into MC1061. | |||
# Check Tecan results | |||
# Prepare PP for MM. | |||
# Re-seed in proper conditions for ON (tecan??) | |||
| | |||
# Tecan worked! | |||
# The LexA rbs variants didn't work. They comp cells I did the library into were clearly just contaminated with broken reporter... | |||
# Picking from Bsrs051 in MC1061 (also picking from comp cells I used yesterday just in case) Picking into 2YT CA | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 14:38, 17 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Wednesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Check Sequencing | |||
# Pick (remember controls!) | |||
# Re-seed for ON and Tecan | |||
# Today, I'm going to make comp cells from Bsrs051 plate. | |||
# I'm also going to transform Bsrs051 (clone 1) into MC1061 to make a new plate. | |||
# I'm also going to pick to make ON culture to make comp cells tomorrow. | |||
# Today I will transform in my Bsrs049 rbs clones into the comp cells as well as Bsrs050. | |||
# I will continue with the Tecan, but not rely on the data. | |||
# Transform Bsrs049's and Bsrs050 into Bsrs051 comp cells | |||
# Transform Bsrs051 into MC1061 | |||
| | |||
# Everything sequenced perfectly except rbs7 which looked like it had a huge insertion at the rbs. So don't use it. | |||
# Plates are f*cked. I need to remake the comp cells... from mini-prep. | |||
# Transformed Bsrs049 (rbs 3, 5, & 8) (forgot Bsrs050!) into both the Bsrs051 comp cells | |||
# Transformed Bsrs051 into MC1061 | |||
# Recovered | |||
# Plated on CAS, plated on CA (also plated plain comp cells on CA and plated comp cells on Spec for contamination check) | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 14:38, 17 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Tuesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Pick | |||
# Mini-Prep, sequence, Transform, plate | |||
| | |||
# Mini-prepped all the new LexA408 rbs members (3-8) | |||
# I transformed all the clone 1's into both Bsrs051 and MC1061, plated on CAS and Spec respectively | |||
# I sent in all clones for sequencing | |||
# I picked two colonies of each constructu: 1) MC1061 2)Bsrs051 (normal) 3) Bsrs051 (broken?) 4-7) Bsrs050, 49.1,49.2,38 in MC1061 8-11) Bsrs050, 49.1,49.2,38 in Bsrs051 comp cells | |||
# These are located in two 24 well blocks because i screwed up the MC1061 constructus (MC1061 are in their own) | |||
# NOTE: Bsrs051 comp cells might be contaminated... but more likely it looks like the promoter 'broke' because it has the CA resistance but is white, but does not grow on Spec alone. That is why I picked both 'normal' (green) and 'broken?' (white) to see growth affects. | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 11:55, 6 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Wednesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Take end-point measurement on my ON from the libraries | |||
# Mini-Prep best, and plasmid separate | |||
# Check Tecan results | |||
| | |||
# Tecan results looked really good; I got a 11-12 fold reduction in GFP with -/+ AHL with 1ul into 200ul. | |||
# Redo these Tecans with sula(408/408) alone in CA, Constructs alone in Spec, Reprter with Dummy plasmid again, and white cells for comparison | |||
# End point measurements showed clones 1, 5, 6, 14, 18, & 24as the lowest normalized RFU. | |||
# I mini-prepped these and they will be of mixed plasmids. | |||
# will do a plasmid separation transformation of all 6 into MC1061 and plate on CA | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 11:55, 6 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Tuesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Check sequencing | |||
# Make Large Scale Comp cells. Come in at 7:30 to Put 5ml of overnight into 500mL of 2YT. | |||
# Pick Library members and grow in CS | |||
# Tecan overnight stuff | |||
| | |||
# Bsrs051-1 was perfect. Bsrs051-2 had a single point mutation that changed a Lysine -> Threonine in the sfGFP. It doesn't seem to have had an effect in the RFU... | |||
# I have to use Bsrs051-2 in the Tecan, but I'll pick from only the Bsrs051.1's ('''next week''') to redo the expeirment. I will also only use Bsrs051-1 for comp cells. | |||
# The library plates looked really good. Nice diversity and a lot of completely off white cells which shows that the double mutant reporter an the LexA408 is working. I picked 24 colonies; 6 from each combination of rbslibrary and reporter. <br> [[File:20120124 13158(crop).png | 400px]]<br> you can see very nice healthy completely white cells as well as healthy green. | |||
# Made large scale comp cells for Bsrs051, and marked them with purple and put them in the drawer second from the bottom | |||
# Also, did an overnight Tecan with LB, DMSO, & DMSO+AHL | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 11:55, 6 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Monday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Mini-Prep reporters and sequence | |||
# Make more comp cells, and digest, ligate, transform in LexA408 library into new reporters. Plate on CAS | |||
# Pick from yesterday's plates, and grow in correct media conditions. | |||
| | |||
# Started re-seed for comp cells and ON to make large scall comp cells tomorrow | |||
# Digested Pcon-C5 and LexA408 rbs library with Bam/xhoi and BgIII/xhoi respectively. | |||
# There are no BsaI sites in my Pcon-lexA408 construct so I can move my system over the analogous system that team cI is using. | |||
# Sent in the mutant mutant promoters for sequencing | |||
# Made double mutant comp cells | |||
# Digest, Ligated, Transformed rbslib.LexA408 into double mutant comp cells | |||
# plate on CAS | |||
# Picked 2 colonies of each transformation (except 2.38 & 2.49.2 because they were redundant and not useful) and grew in LB CS | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 11:01, 8 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Easter Sunday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Re-seeding to mini-prep and sequence confirm tomorrow | |||
# Making comp cells from both Bsrs051 reporters | |||
# Transforming in Full-LexA408 & LexA-TraR | |||
# Recover | |||
# Plate on CAS | |||
| | |||
# Re-seeded the Bsrs051 ON's to make new ON and to make Comp cells | |||
# Mini-prepped (will sequence tomorrow/ in Scott Box Under Construction) | |||
# Made comp cells from Bsrs051 clone 1 and 2 | |||
# Doing four transformations into each. Bsrs038, Bsrs49.1, Bsrs049.2, and Bsrs050. Should be green, white, white, and green respectively. | |||
# All will be plated on CAS | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 11:01, 8 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Saturday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Pick | |||
| | |||
# Picked two green colonies into CA | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 11:55, 6 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Friday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Analyze Sequencing | |||
# Eco/Bam DIgest PCR product of new reporter | |||
# Eco/Bam 1601CA maybe some other 1601... 1601c? 1601a? | |||
# Gel Purify | |||
# Ligate | |||
# Transform | |||
# Plate | |||
| | |||
# multiple nhe1 sites explain weird ass bands from yesterday. Luckily one of the LexA-TraR constructs ligated correctly So it can be used. | |||
# (Reminding myself to ligate the rbslib.LexA with Ptet and no-nhe1 Pcon's) | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 08:41, 5 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Thursday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Mini-Prep | |||
# Map, Sequence | |||
| | |||
# There was only one red culture of 3 for both Trunc5 and Truc17. The background (Ptet-cI-rbsRFp) should be red, and so should mine since its on a Pcon, so there should be no white colonies. Strange... checking band sizes. My LexA construcuts should be 200 bps less than the cI constructs. (TraR being ~1000 & 1200; RFP being around 1150 & 1350) <br> [[File:20120406 14110(small).png |100px]] | |||
# Clearly some mixed ligations, but I'm sequencing 1, 2, 5, 6, 8, 10 & 11. | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 08:26, 4 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Wednesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Pick | |||
| | |||
# Picked 3 colonies each into LB Spec | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 08:26, 3 April 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Tuesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Check digest band sizes | |||
# ((((BgIII/xhoI Digest library members of rbs.LexA408 # Bam/xhoI Digest 9525-Pcon; wait to have 408/408 reporters)))) | |||
# Nhe1/Eco 9525-Bsrs049 PCR Truncations | |||
# Nhe1/Eco TraR, LambdaRep, MukF, ER? (pick large/plasmid band) | |||
# Gel Purify | |||
# Ligate | |||
# Transform | |||
# Recover, Plate | |||
| | |||
# Mini-prepped some 9525 cI fusions for Nina; then digested the TraR fusion and RFP drop out fusion | |||
# Digested: <br> [[File:20120404 14080(small).png|100px]] | |||
# It looks faint because the Eth. Bromide was old, but should be high concentration. | |||
# Started Ligation of T5 & T17 with both TraT and rbs.RFP | |||
# Transformed into MC1061 and plated on Spec | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 10:48, 21 March 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Wednesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "1000"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
|- valign="top" | |||
| | |||
# MM notes: | |||
# create a 408/408 reporter | |||
# Test a homo system with LexA408-TraR (this will be easy and should take about a week) & see if you can get AHL dependancy | |||
# In meantime see if you can just KO LexA (cell viability) '''(LexA KO's are not viable. they will not grow.)''' | |||
# Also, make sure that my system isn't demonstrating OD growth defects or toxicity; Ask gabe/postdocs how to gauge for toxicity | |||
# Get off Spec Plasmids (do in parallel in 9145 or something, not 9525); change reporter to just Cam... maybe? Anything except Spec, and not Amp if we're doing 9145... (Use K, C, & A) | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 10:48, 21 March 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Spencer is on Spring break 3/22-4/2<hr></font></div> | |||
==[[User:SpencerScott|SpencerScott]] 10:48, 21 March 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Wednesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Mini-Prep the Bsrs049's and QC | |||
# Scrape library members; Mini-Prep | |||
# Ask Robin & Austin to Mini-Prep more 9525-1834 / Two vials please! | |||
# DESIGN OLIGOS (over spring break; study for your exam first) | |||
| | |||
# Mini-Prepped the Bsrs049's and the scraped libraries. | |||
# Sequenced well (throw away 1.1 and 2.1) 2.21 is the same as 2.17 but lacks the silent mutation so might be better to use. really shouldnt matter though. | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 16:29, 20 March 2012 (PDT)== | |||
<div style="text-align: center; background: #AFF0D8"><font size="5" face="Georgia" color="black"><br>Tuesday<hr></font></div> | |||
{| cellpadding="2" style="background: #FFFAE3; color: black" | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Plan</font> | |||
! width = "500"| <font size="4" face="Georgia" color="black">The Day</font> | |||
|- valign="top" | |||
| | |||
# Check QC | |||
# Pick two colonies (white!); grow in spec. | |||
# PCR for truncation | |||
# PCR cleanup | |||
# Digest | |||
# Ligate | |||
# Transform; Plate | |||
# Design oligos to give normal LexA the same rbs | |||
# Design Oligos for KO's and such. | |||
| | |||
# Everything was good except the clone 1 which was really weird. | |||
# Picked two colonies of each and grew in Spec | |||
# Did some PCR's off the 5 and 17 clone for truncations and a re-do of the rbs library | |||
# the Truncations failed because I used to wrong digestion; its okay because I can make these in parallel with the w.t. LexA later | |||
# The rbs library's are gel purified and are ligating into 9525 | |||
# Will Transform into MC1061, recover, and Plate with beads on Spec | |||
|} | |||
==[[User:SpencerScott|SpencerScott]] 10:29, 19 March 2012 (PDT)== | ==[[User:SpencerScott|SpencerScott]] 10:29, 19 March 2012 (PDT)== | ||
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# ganna pick 24 and do Col PCR on them all | # ganna pick 24 and do Col PCR on them all | ||
# There were 126 colonies in the 1:1000 dilution and 90ul of plating. | # There were 126 colonies in the 1:1000 dilution and 90ul of plating. | ||
# Ran Col PCR Ran on Gel: All worked except 2 that had mixed bands!<br> [[Image:20120314 13906(small).png|100px]] [[ | # Ran Col PCR Ran on Gel: All worked except 2 that had mixed bands!<br> [[Image:20120314 13906(small).png|100px]] [[Image:20120314 13907(small).png|100px]] | ||
|} | |} | ||
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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. | ||
|} | |} |
Latest revision as of 11:02, 23 April 2012
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SpencerScott 11:01, 8 April 2012 (PDT)
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SpencerScott 10:48, 21 March 2012 (PDT)
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SpencerScott 10:48, 21 March 2012 (PDT)
Spencer is on Spring break 3/22-4/2
SpencerScott 10:48, 21 March 2012 (PDT)
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SpencerScott 15:00, 6 March 2012 (PST)
Spencer Is in New Zealand 2/24-3/6
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