SBB12Ntbk-SpencerScott

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SpencerScott 10:29, 19 March 2012 (PDT)


Monday
The Plan The Day
  1. Re-seed clones 1, 5, 17, 21, and controls for ON
  2. Re seed same clones for Tecan
  3. Mini-Prep said clones
  4. Send for Sequencing with special primers
  5. Design Oligos for KO's and such.


SpencerScott 10:27, 18 March 2012 (PDT)


Sunday
The Plan The Day
  1. Re-seed for more overnights
  2. Re-seed into Tecan
  3. 24 Mini-preps
  4. Send for QC! with our special oligos
  5. Plasmid separate somehow?
  6. Maybe transform all of them even though we don't know QC stuff
  1. Re-seeded for more overnights
  2. Re-seeded to set up a Tecan
  3. Mini-prepped only clone 1 & 5 because they looked promising
  4. Will send for sequencing on Monday

SpencerScott 10:25, 17 March 2012 (PDT)


Saturday
The Plan The Day
  1. Pick 24 white cells
  2. Run colony PCR?
  3. Make/Use new CAS media
  1. Picked 24 colonies and grew in CS

SpencerScott 11:59, 16 March 2012 (PDT)


Friday
The Plan The Day
  1. Scrape
  2. Mini-prep
  3. Digest
  4. Ligate with Pcon's
  5. Transform into Bsrs048 comp cells! (Remember Controls!, dummy 9525 plasmid on CAS and regular comp cells on CA)
  6. Plate on CAS
  1. Scraped but only used 1/4 of the pellet to make the mini-preps
  2. Digested Pcon (clone 2 & 3) with Bam /xhoi (1480+1000, S)
  3. Digested Bsrs038 with rbs lib with BgIII/xhoi (2100 + 900, L) (could definately see background bands from 1834, but I think I got the right bands. Background will be at 3300 & 900)


SpencerScott 11:59, 15 March 2012 (PDT)


Thursday
The Plan The Day
  1. Digested PCR's
  2. Purified, & Ligated into 9525
  3. Transformed and Plated on Spec
  1. Digested PCR's
  2. Purified, & Ligated into 9525
  3. Transformed and Plated on Spec

SpencerScott 13:50, 11 March 2012 (PDT)


Wednesday
The Plan The Day
  1. Re-seed for more overnights
  2. Re-seed into Tecan
  3. 24 Mini-preps
  4. Send for QC! with our special oligos
  5. Plasmid seperate somehow?
  6. Maybe transform all of them even though we don't know QC stuff
  1. Need to re-think plan. I have to KO both LexA and sulA/sulA promoter!
  2. In the meantime I might just carry on with cloning to make the mutants and truncations...
  3. Nevermind! I can get around this and use the genomic LexA to my advantage in the Bsrs038 strains.
  4. Started SOEing on 9525-rbslib-LexA constructs to make the 408 mutant so that i can transform them into Bsrs038 cells.
  5. Did two rounds of SOEing. I ran it on a gel and the bands look good but could be brighter so I'm doing what Nina suggested and doing one more round of PCR just to amplify the final product. I will pick that up tomorrow.

SpencerScott 13:50, 11 March 2012 (PDT)


Tuesday
The Plan The Day
  1. Pick white colonies! (if the controls check out)
  2. Make more CAS plates
  1. Not as white as I would hope for but compared to normal white cells some are alright.
  2. ganna pick 24 and do Col PCR on them all
  3. There were 126 colonies in the 1:1000 dilution and 90ul of plating.
  4. Ran Col PCR Ran on Gel: All worked except 2 that had mixed bands!

SpencerScott 10:07, 12 March 2012 (PDT)


Monday
The Plan The Day
  1. Bam/Xhoi Digest 9525-C5
  2. BgIII/Xhoi Digest 9525-(rbs.lib)-bsrs038
  3. Purify
  4. Ligate Two separate batches
  5. Transform into MC1061 & SulA reporter strains
  6. Include controls
  7. Plate on Spec & CAS
  1. Started large scale comp cell prep of Bsrs047 and Bsrs048 in 1601CA
  2. Digested Pcon (clone 2 & 3) with Bam /xhoi (1480+1000, S)
  3. Digested Bsrs038 with rbs lib with BgIII/xhoi (2100 + 900, L) (could definately see background bands from 1834, but I think I got the right bands.)
  4. Ligated
  5. Transformed into my new Bsrs037 comp cells. With a 9525-Pcon dummy control. Also plated the comp cells just on Spec to check for contamination.
  6. Recovering
  7. Plated 30ul of 1x, 90ul of 1/100, & 90ul of 1/1000
  8. Need to make more CAS plates

SpencerScott 13:50, 11 March 2012 (PDT)


Sunday
The Plan The Day
  1. Pick
  1. Everything sequenced well (except one Pcon) Good to go!

SpencerScott 11:41, 10 March 2012 (PST)


Saturday
The Plan The Day
  1. Mini-Prep
  2. Map; Sequence
  3. Sequence Pcons
  4. Transform; Plate
  1. One of each SulA reporter clone didn't grow. The ones that did grow weren't very high OD. I'm going to pick more if the mapping doesn't look good.
  2. Mini-prep'd
  3. E/B digested bsrs047/048 will map
  4. Transformed bsrs047/048 will plate on CA
  5. Sent all the Pcons and bsrs047 & bsrs048 in for sequencing
  6. Reporters mapped well.

SpencerScott 13:51, 9 March 2012 (PST)


Friday
The Plan The Day
  1. Pick
  2. Mini-Prep
  1. Scrape both plates of the 9525-(rbs.lib)-Bsrs038 only in the concentrated parts
  2. Ask Gabe how to do it again…
  3. Mini-Prep


SpencerScott 15:00, 6 March 2012 (PST)


Thursday
The Plan The Day
  1. Change everything
  2. Transfer reporters to 1601CA
  3. Start construction of Pcon-rbs-LexA
  1. E/B Digested Bsrs047 & Bsrs048 and 1601CA.
  2. Gel Purify smaller bands
  3. Ligate Bsrs047 and Bsrs048 into 1601CA
  4. Ligate Pcon-C5 into your pre-digested 9525
  5. Transform into MC1061
  6. Plate on CA and on Spec respectively

SpencerScott 15:00, 6 March 2012 (PST)


Wednesday
The Plan The Day
  1. Make Bsrs047 and Bsrs048 Comp Cells
  2. EIPCR rbs's onto my two LexA constructs
  3. Map, E/B Digest & E/B digest 9525 dropout or backbone, Map
  4. Purify
  5. Ligate
  6. Transform into MC1061; also transform into SulA comp cells
  1. Sequencing was perfect for all reporter constructs
  2. Started Bsrs047 and Bsrs048 comp cell cultures in 2YT-Spec at 8:48am
  3. 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)
  4. Digested; Picked lanes 1, 3, 5, 7 & 8
  5. Purified, Ligated 1 with 8 and 5 with 7
  6. Transformed, Recovered, Plated on Spec full plates

SpencerScott 15:00, 6 March 2012 (PST)


Tuesday
The Plan The Day
  1. Send in mini-preps for sequencing
  2. transform in mini-preps to create comp cells
  1. Sent for sequencing; Trasnformed all 4 into MC1061 and plated on Spec

SpencerScott 15:00, 6 March 2012 (PST)


Spencer Is in New Zealand 2/24-3/6


SpencerScott 14:59, 6 March 2012 (PST)


Friday
The Plan The Day
  1. Pick/ Grow
  2. Ask someone to Mini-prep on Saturday
  1. Gabe mini-prepped for me

SpencerScott 10:18, 23 February 2012 (PST)


Thursday
The Plan The Day
  1. Check Sequencing
  2. If oligos come in, ask how long PCR reaction stay good; and PCR LexA constructs to add rbs's
  3. Also see how long colonies on a plate last, and potentially digest, ligate, transform, plate
  1. Great sequencing! Got all of our parts
  2. Bam/xhoi& BgIII/xhoi digested 1600-Bsrs045/Bsrs046& 1600-jtk2541:
  3. (1486, 1227, S) & (2239, 1149, L)
  4. Ligated; Transformed into MC1061; Recovered; Plated on Spec

SpencerScott 20:37, 22 February 2012 (PST)


Wednesday
The Plan The Day
  1. mini-prep, map, sequence
  2. BgIII/Brick Assembly of SulA reporters with JTK2541
  3. PCR LexA constructs to add rbs's!
  1. The shaker wasn't at 37... so nothing grew up. Picking into 2YT and will mini-prep in ~6-8 hours.
  2. 24 mini-preps!
  3. 24 Eco/Bam Digestions!
  4. Lengths: 1-4: (2635, 753); 5-10: (2066, 615); 11-16: (2481, 615); 17-24: (2481, 1108)
  5. They all worked except the last one. Sent in for sequencing 1, 2, 8, 9, 10, 11, 12, 14, 17, 19, 21, & 22.

SpencerScott 14:02, 21 February 2012 (PST)


Tuesday
The Plan The Day
  1. Ligate JTK2541 into 1600 & 1601CA; Ligate LexAFull(w.t) into 9145 & 9525
  2. Ligate both LexA(1-87)(w.t.)'s into 9525-nhe1.rbs.RFP
  3. Transform into MC1061, Recover, Plate on Spec, CA, Amp, Spec, Spec respectively
  4. Come in late at night to pick Colonies! Grow in respective media. ~12 hours later
  1. Five Ligations:
    (1) JTK2541&1600
    (2) LexAFull & 9145
    (3) LexA Full & 9525
    (4) LexA(1-87).1 in 9525
    (5) LexA(1-87).2 in 9525
  2. Transformed everything into MC1061, recovered, plated.

SpencerScott 15:19, 20 February 2012 (PST)


Monday
The Plan The Day
  1. Map PCR cleanup's of LexA (before and after digestion)
  2. Re-do Ligations of PCR cleanup's of LexA into 9145 & 9525; maybe try a p15? And 1600
  3. Do we need to do chasis modifications?
  4. Eco/Bam JTK2541 into 1600 and 1601CA
  1. The PCR's of LexA look good. I will try to re-digest and re-ligate them.
  2. Need to move JTK2541 into 1600 and 1601CA so I can ligate it with SulA promoters.
  3. Eco/Bam, JTK2541, 1600, 1601CA, 9145, 9525, & LexA (w.t)
    (1) (2481+753, S)
    (2) (2635+78, L)
    (3) (3205+78, L)
    (4) (2066+342, L)
    (5) (2481+1854, L)
    (6) (615)
  4. Eco/Nhe1 LexA(1-87)'s and 9525-1834
    (1) 261
    (2) 261
    (3) (3313+1022, L)
  5. Gel Purify

SpencerScott 15:22, 19 February 2012 (PST)


Sunday
The Plan The Day
  1. Check Sequence Results
  2. Do some EIPCR on LexA w.t. (and maybe the trunc if it sequenced well!)
  3. PCR cleanup
  1. The SulA Promoters assembled in both 1601CA and 1600. However, none of the LexA constructs worked. They were all really strange.
  2. Sequence the LexA PCRs??
  3. Need to somehow get LexA!

SpencerScott 12:33, 18 February 2012 (PST)


Saturday
The Plan The Day
  1. Check Sequence Results
  2. mini-prep SulA promoter cells;sequence
  1. Sequences sucked. The assembly went wrong, somehow only the stress-ToxR was left in most of them. Bss003 and Bss006 sequences well at least. Sent the other potential Bsrs044 constructs in for sequencing along with 14 other sulA promoter sequences and some LexA w.t sequences.

SpencerScott 12:33, 17 February 2012 (PST)


Friday
The Plan The Day
  1. Re-seed into Arabinose titration Tecan and run overnight.
  2. QC 9145-Bsrs041 cells with pBgl00001-Bsrs043 & pBgl00001-Bss006 (mini-prep them)
  3. Mini-prep 9525-LexA constructs; Map; Sequence
  4. Pick SulA promoter cells; grow in CA; Pick 9145-Bsrs038 cells (not red!) grow in AMP
  5. Re-Sequence Bss003 & Bss006
  1. Picked 4 colonies for each SulA promoter grew in CA for the 1601CA and Spec for the 1600.
    Also picked four non-red 9145-Bsrs038 cells, grew in AMP.
  2. Set up the titrations
  3. Eco/Bam'd the 8 Lex(1-87) to map. (desired size: 2400,1100)

    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.
  4. Sent for sequencing 1, 2, 3, 5, 8, Bss003, Bss006, and the mixed plasmids reading from ca56

SpencerScott 11:17, 16 February 2012 (PST)


Thursday
The Plan The Day
  1. Pick from 9145-Bsrs041 cells with pBgl00001-Bsrs043 (Grow in AT) and with 9525-Bsrs043 (Grow in AS)
  2. Check Sequencing
  3. Pick from 9525-LexA constructs (Grow in Spec)
  4. Wobble PCR SulA promoter (op+/op+) & (408/op+)
  5. Short Frag Clean-Ups
  6. Eco/Bam Digests on PCR's and 1601CA
  7. Gel Purify
  8. Two Ligations
  9. Two Transformations, Recover, Plate on CA
  1. Wobbled 189f/190r & 191f/192r (~100bps)
  2. Small Frag clean-up done.
  3. Eco/Bam'd the two Wobble reactions and 1601CA-Bss52.
    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.
  4. Sequencing data came back; I forgot to specify primers so the only read I got was ca56 off Bss006 which looked great.
  5. Gel purified (small frag purified for the sula promoters)
  6. 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)
  7. Transformed into MC1061; Recovered for 30 mins in 2YT
  8. Plated 1601CA on CA and 1600 on Spec.
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