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| | {{:Team:Arizona_State/Templates/sidebar|title=Notebook: July}} | | {{:Team:Arizona_State/Templates/sidebar|title=Notebook: July}} |
| - | __NOTOC__
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| - | == Friday, July 1 ==
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| - | * Ran a gel of PCRed CAS genes from last night
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| - | * Transformation of NEB cells:
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| - | :* DA: Kan x2, LB
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| - | :* DB: Kan x2, LB
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| - | :* Seq1: Kan x2
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| - | :* CMR: Kan x2, LB
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| - |
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| - | == Saturday, July 2 ==
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| - | * Plates from Friday: successful transformation for all except CMR
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| - | :* Need to find a plasmid that can handle such a large insert
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| - | :* Made overnight cultures of colonies
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| - | * Ligation, transformation of RA, RB onto kan plates
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| - | * Gels of more PCR products: no usable bands
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| - |
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| - | == Tuesday, July 5 ==
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| - | * Another PCR attempt with new CAS primers (A, C)
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| - | * Miniprep of DA, DB, RA, SEQ1
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| - | * Restriction digest of:
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| - | :* SEQ1, DA, RA, (ES, EX) in PSB1K3
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| - | * Tried 2 different ligation protocols:
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| - | :* Normal protocol:
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| - | ::* seq1 + seq1, da + da, ra + ra
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| - | ::* transformation of ligation products, plated on kan (NEB cells)
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| - | :* Gingko protocol:
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| - | ::* seq1 (ES, XP) + PSB1A3 (EP)
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| - | ::* transformation, plated on amp (NEB cells)
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| - | * glycerol stocks of seq1, da, db, ra, rb in PSB1K3
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| - |
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| - | == Wednesday, July 6 ==
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| - | * Transformation results:
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| - | :* Kan plates:
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| - | ::* all successful (?) (seq1 + seq1, da + da, ra + ra), will be sequenced (ordered biobrich forward / reverse primers)
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| - | ::* made liquid culture for miniprep tomorrow
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| - | :* Amp plates:
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| - | ::* no success
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| - | * PCR:
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| - | :* cas_b, cas_c both unsuccessful (see pictures of gels)
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| - | :* cas_d run, will visualize tomorrow
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| - | :* adjusting settings for cas_e_f, cas_3_r: 2 parallel
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| - | ::* touchdown to 70 using builtin touchdown
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| - | ::* constant 70
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| - | :* probably need new primers
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| - |
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| - | == Thursday, July 7 ==
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| - | * Miniprep of:
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| - | :* DADA (kit buffer) in PSB1k3
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| - | :* RARA (kit buffer) in PSB1k3
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| - | :* Seq1Seq1 (kit buffer, Dan's buffer) in PSB1k3
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| - | :* Consensus: Dan's buffer that he created for lysis works
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| - | :* Nanodrop results: (DADA 48 ng/uL, RARA 68 ng/uL, Seq1Seq1 Dan 73.5 ng/uL, Seq1Seq1 Kit 98 ng/uL)
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| - |
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| - | * Gel of "homerun" attempt from yesterday, Block A touchdown + Block B constant 70 degC
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| - | :* Unsuccessful, deformed gel near top, nonspecific binding, weird band at ~800bp (see photo)
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| - |
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| - | * Met w/ 3 grad students (Kurt, Jack?, Bo) and had good discussion
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| - | :* Gel techniques: doing gels in 4 degree room, using X-tracta gel removers for gel band extraction
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| - | :* Assembly: problems with large insertions (see Infusion)
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| - | :* Infusion (clonetech)
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| - |
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| - | * Beginning to construct our GFP test plasmid:
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| - | :* Transformation of biobrick promoter J23101 onto amp (2 plates)
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| - | :* Streak plated E0840 GFP part onto amp from glycerol stock (3 plates)
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| - |
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| - | * Determined theoretical 3 part PCR (casEDC, casBA, case)
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| - |
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| - | * PCR attempts (see photo):
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| - | :* CasE forward, Cas3 reverse again but w/varying template DNA concentrations
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| - | ::* 225 ng/uL, 112.5 ng/uL, 56 ng/uL (previous starting conc. was 450, which is too high and could have caused problems)
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| - | ::* started @ 80deg, increment -.3
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| - | :* Cas3 forward/reverse w/same varying template DNA concentrations
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| - |
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| - | * Restrictions:
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| - | :* Seq1Seq1 (ES, EX)
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| - | :* DADA (ES, EX)
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| - | :* RARA (ES, EX)
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| - |
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| - | * Ligation for theoretical 4-mer:
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| - | :* 2x Seq1Seq1
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| - | :* 2x DADA
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| - | :* 2x RARA
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| - | :* Plated onto 3 kan plates using NEB cells.
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| - |
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| - | == Friday, July 8 ==
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| - | * Yesterday's plates:
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| - | :* promoter transformation: no colonies
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| - | :* GFP streak plate: usable colonies
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| - | :* DAx4, RAx4, seq1 transformation: usable colonies
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| - | * Got order from IGEM (psb1a3 and agar stab of J04430)
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| - | :* transformed psb1a3 plasmid to make stock
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| - | :* streak plated agar stab of J04430
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| - |
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| - | == Saturday / Sunday, July 9 / 10 ==
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| - | * Gel of theoretical array (1x, 4x):
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| - | :* plasmid backbone visible, but no inserts
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| - | :* will need to start over from 1x
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| - | * PCR: homerun, EDC, AB, 3
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| - | :* no success
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| - |
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| - | == Monday, July 11 ==
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| - | * Transformation results from Friday:
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| - | :* PSB1A3: DNA contamination? Red colononies as well as normal growing on plates
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| - | ::* will sequence once we get biobrick standard primers
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| - | :* agar stab of part J04430: normal (glowing)
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| - |
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| - | * made liquid cultures of:
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| - | :* SEQ1, DA, RA (8x) in PSB1k3
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| - | :* PSB1A3 (pink, white colonies)
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| - | :* J04430
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| - |
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| - | * restriction:
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| - | :* restarting from 1x in PSB1K3
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| - | :* gingko:
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| - | ::* seq1: es, xp
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| - | ::* da: es, xp
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| - | ::* ra: es, xp
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| - | ::* e0840: ep to get PSB1A3 backbone
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| - | :* normal:
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| - | ::* seq1: es, ex
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| - | ::* da: es, ex
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| - | ::* ra: es, ex
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| - |
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| - | * ligation:
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| - | :* 2x seq1, da, ra 2 ways:
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| - | ::* psb1k3
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| - | ::* psb1a3 (gingko)
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| - |
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| - | * transformation onto 14 plates:
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| - | :* new promoter (g18 on plate 1) in duplicate
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| - | :* ligation products in duplicate
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| - |
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| - | * planning:
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| - | :* planned assembly of plasmid construct (cas genes, leader sequence, array) using duet plasmid
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| - |
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| - | == Tuesday, July 12 ==
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| - | * Transformation results: all successful (14 / 14)
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| - |
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| - | * liquid cultures of:
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| - | :* promoter
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| - | :* e0840 (gingko, other)
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| - | :* 2x seq1, da, ra
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| - |
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| - | * Miniprep using new ethanol protocol:
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| - | :* 8x (ra, da, seq1)
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| - | ::* nanodropped, yields: 50-1000 ng / ul (hooray)
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| - | :* psb1a3 red colony
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| - |
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| - | * sequencing orders:
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| - | :* 1x, 2x, 4x (3 times each)
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| - |
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| - | * submitted primers / synthesis order for our final design:
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| - | :* new cmr
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| - | :* new cas3
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| - | :* new casabcde
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| - | :* adapter plasmids for leader sequences for cmr, cas
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| - | :* ordered new restriction enzymes
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