Team:Arizona State/Notebook/August

 

Monday, August 1

• Checked plates

• Nothing

• Another RLT

• This time with a vengeance
• 2x Seq1, RA, DA in 1K3
• TSS cells

• Made more LB Kan broth

• Because previous attempt was bad, ampR grew in it

• Tested TSS cells with puc19
• Submitted BMES abstract

Tuesday, August 2

• transformation results from yesterday:

• 4 colonies on seq1
• DA colonies all clumped together
• RA fail, among stacks of failed transformations 3:

• new RLT for 2x

• Varying DNA volumes added (1, 2.5, and 5 uL RLTs)

• PCR:

• seq1, RA, DA in puc57 using puc57 primers

• pcr settings: from previous

• Gel results: look the same as before

• clear bands ~100 for Seq1
• ~200 for DA, RA

• submitted for sequencing:

• BAHHH jason
• Resubmitted 1x Seq1, RA, DA(pcr and prep), GFP, RFP, Cas3

• Streak plates

• BL21 DE3 (x2, one clearly better than the other)
• 2xDA in pSB1K3 (clumped colonies initially)

• Liquid culture

• 2xSeq1 in psb1k3
• BL21DE3 from glycerol stock

• test tube with larger stopper may be contaminated

Wednesday, August 3

• Liquid cultures

• 2x Seq1
• 1xDA

• PCR

• cleanup of PCR amplified Seq1, RA, DA from puc57
• (we gelled this last night, looked good)
• Used RA from this for RLT -->

• RLT

• RA from PCR cleanup

• Prepared TSS competent cells
• Notes:

• Decided not to continue with p. furiosus due to the cmr array's length (9kb)
• Instead, we decided to explore our other options, like Type II streptococcus strains (which only have one cas protein involved in their mechanisms of silencing)

Thursday, August 4

• ABCDE PCR with the newest primers (v3)

• 98deg initial denaturation for 30 seconds
• cycle {10 sec at 98 degrees

• anneal at 63 degrees for 30 sec
• elongate at 72 degrees for 130 sec}

• 35 cycles
• then extension for 450 seconds

• Results show clearly a band above where we would like it, at around 8kbp, and also some bands below the desired length at around 2kbp

Friday, August 5

• miniprep:

• using old protocol
• RAGE, SEGE, RFP overnight cultures

• gel:

• PCR from last night

• casABCDE (upper):

• see photo
• band > 10k, genomic?

• casABCDE (lower):

• same

• gel extraction of PCR 8-4 (see photo)
• PCR of gel extraction CasABCDE:

• sample 1,2,3

• Primers: CasABcDE_V3 F/R

• Thermocycle: ABCDE802
• Template: gel extraction sample 1 of PCR 8-4 (above)

• Mg2+ gradient

• sample1: 0 mM
• sample2: 1mM
• sample3: 2mM
• Cycle strarted 5:30pm

• Colony PCR:

• sample 1c, 2c,3c
• Primers: CasABcDE_V3 F/R

• Thermocycle: ABCDE802

• Template: colonies from Jon's source plate

• 1c:BL21 DE3 -control
• 2c: BL21 DE3 -control
• 3c: Mg1655 +(hopefully we can amplify cas genes this way)

• Cycle started 5:50pm

• New 10mM dNTP solution made from dNTP stock.
• RLT:

• todo

Monday, August 8

GROUP MEETING

• Kylie's Update

• Tried SLIC, not very successful

• (SLIC is a modification of Gibson)

• New Ligation Method

• Idea is to cut with X, S
• This can then stick together multiple times, theoretically
• Different ligation times can allow for longer arrays (1x, 2x, 3x, 4x, 5x…)
• *Tried this for Seq1

• Result (pic in Dropbox): multiple bands at increasingly large lengths

• w/normal ligation control to compare

• Consideration: could ligate "upside-down" - will this have any negative effect?

• Approach

• RB + RA --> RSR or "Seq2" essentially

• Then, we can ligate using the KSB method

• Question: if there is an "upside-down" section, what even happens?

• Is the complement side just skipped over? Will it hairpin? Will it mess something up?
• Our best guess is that it will be fine, perhaps not every spacer will "work" but enough will

• What about self-targeting?

• Sci tag prevents self targeting

• Organization

• Personal Dropbox Lab Notebooks

• Specialization

• Dan -- RB + RA, b. halodurans focus
• Abhi -- "Seq2" assembly (XS method, etc)
• Kylie -- Listeria innocua
• Madeline -- CasABCDE, Cas3 (and put in Duet)
• Keith -- Ginkgo
• Nisarg -- Ginkgo
• Ryan -- CasABCDE, Cas3
• Ruben -- drylab
• Ethan -- drylab
• Joseph -- XS method (seq1, RA)

• To order

• Gloves
• Plates
• t4 polymerase
• miniprep
• listeria innocua items

Tuesday, August 9

Wednesday, August 10

Thursday, August 11

Friday, August 12

Saturday, August 13

Sunday, August 14

Monday, August 15

Team meeting

• Updates

• Cas genes - not looking good, ran out of Phusion, will keep trying w/ some adjustments
• Ginkgo woes

• Today

• Liquid culture of MG1655

• to make it competent

• Sequencing

• Leader Sequences
• 2x RA
• Duet
• Whatever else is on the board

• Streak plate pRSF Duet (old mini prep, could be weird)
• New media, new plates
• Streak plate polySeq1

• Liquid culture later, mini prep tonight or tomorrow

• Cas3 PCR w/ uber long elongation

• Look again at annealing temps

Tuesday, August 16

• Liquid cultures

• Kylie's 4L1A3 (orig. XS ligation of Seq1 in pSB1A3
• 1655 from glycerol stock for genome prep and competency
• BL21 DE3 for more TSS cells
• Keith's 2x RA and Seq1 streak plates from 8/15 (grew this time!)

• used LB-Kan broth from 8/11 (made by Abhi)

• PCR amplification of Seq1 in pUC57

• Gel visualized, ~150bp bands in samples 4,8,10

• still on transilluminator

• samples labeled and in -20

• PCR log updated
• New PCR of Cas genes

• Going to try Cas3 with 7/24 settings with OLD PRIMERS
• Also, try an ABCDE Nest with 8x MgCl2 (this was good before) and a small temperature gradient

• We can then use the result as a template (and perhaps sequence it for funzies to see if we're getting it)

• ((more details in Madeline's folder, including results, though pic is here too))

Wednesday, August 17

• made tss cells (bl21, mg1655)
• genome prep: mg1655

Thursday, August 18

Friday, August 19

Saturday, August 20

Sunday, August 21

Monday, August 22

Tuesday, August 23

Wednesday, August 24

Thursday, August 25

Friday, August 26

Saturday, August 27

Sunday, August 28

Monday, August 29

Tuesday, August 30

== Wednesday, August 31 ==