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Notebook: July

Friday, July 1

• Ran a gel of PCRed CAS genes from last night
• Transformation of NEB cells:

• DA: Kan x2, LB
• DB: Kan x2, LB
• Seq1: Kan x2
• CMR: Kan x2, LB

Saturday, July 2

• Plates from Friday: successful transformation for all except CMR

• Need to find a plasmid that can handle such a large insert
• Made overnight cultures of colonies

• Ligation, transformation of RA, RB onto kan plates
• Gels of more PCR products: no usable bands

Tuesday, July 5

• Another PCR attempt with new CAS primers (A, C)
• Miniprep of DA, DB, RA, SEQ1
• Restriction digest of:

• SEQ1, DA, RA, (ES, EX) in PSB1K3

• Tried 2 different ligation protocols:

• Normal protocol:

• seq1 + seq1, da + da, ra + ra
• transformation of ligation products, plated on kan (NEB cells)

• Gingko protocol:

• seq1 (ES, XP) + PSB1A3 (EP)
• transformation, plated on amp (NEB cells)

• glycerol stocks of seq1, da, db, ra, rb in PSB1K3

Wednesday, July 6

• Transformation results:

• Kan plates:

• all successful (?) (seq1 + seq1, da + da, ra + ra), will be sequenced (ordered biobrich forward / reverse primers)
• made liquid culture for miniprep tomorrow

• Amp plates:

• no success

• PCR:

• cas_b, cas_c both unsuccessful (see pictures of gels)
• cas_d run, will visualize tomorrow
• adjusting settings for cas_e_f, cas_3_r: 2 parallel

• touchdown to 70 using builtin touchdown
• constant 70

• probably need new primers

Thursday, July 7

• Miniprep of:

• DADA (kit buffer) in PSB1k3
• RARA (kit buffer) in PSB1k3
• Seq1Seq1 (kit buffer, Dan's buffer) in PSB1k3
• Consensus: Dan's buffer that he created for lysis works
• Nanodrop results: (DADA 48 ng/uL, RARA 68 ng/uL, Seq1Seq1 Dan 73.5 ng/uL, Seq1Seq1 Kit 98 ng/uL)

• Gel of "homerun" attempt from yesterday, Block A touchdown + Block B constant 70 degC

• Unsuccessful, deformed gel near top, nonspecific binding, weird band at ~800bp (see photo)

• Met w/ 3 grad students (Kurt, Jack?, Bo) and had good discussion

• Gel techniques: doing gels in 4 degree room, using X-tracta gel removers for gel band extraction
• Assembly: problems with large insertions (see Infusion)
• Infusion (clonetech)

• Beginning to construct our GFP test plasmid:

• Transformation of biobrick promoter J23101 onto amp (2 plates)
• Streak plated E0840 GFP part onto amp from glycerol stock (3 plates)

• Determined theoretical 3 part PCR (casEDC, casBA, case)

• PCR attempts (see photo):

• CasE forward, Cas3 reverse again but w/varying template DNA concentrations

• 225 ng/uL, 112.5 ng/uL, 56 ng/uL (previous starting conc. was 450, which is too high and could have caused problems)
• started @ 80deg, increment -.3

• Cas3 forward/reverse w/same varying template DNA concentrations

• Restrictions:

• Seq1Seq1 (ES, EX)
• DADA (ES, EX)
• RARA (ES, EX)

• Ligation for theoretical 4-mer:

• 2x Seq1Seq1
• 2x DADA
• 2x RARA
• Plated onto 3 kan plates using NEB cells.

Friday, July 8

• Yesterday's plates:

• promoter transformation: no colonies
• GFP streak plate: usable colonies
• DAx4, RAx4, seq1 transformation: usable colonies

• Got order from IGEM (psb1a3 and agar stab of J04430)

• transformed psb1a3 plasmid to make stock
• streak plated agar stab of J04430

Saturday / Sunday, July 9 / 10

• Gel of theoretical array (1x, 4x):

• plasmid backbone visible, but no inserts
• will need to start over from 1x

• PCR: homerun, EDC, AB, 3

• no success

Monday, July 11

• Transformation results from Friday:

• PSB1A3: DNA contamination? Red colononies as well as normal growing on plates

• will sequence once we get biobrick standard primers

• agar stab of part J04430: normal (glowing)

• made liquid cultures of:

• SEQ1, DA, RA (8x) in PSB1k3
• PSB1A3 (pink, white colonies)
• J04430

• restriction:

• restarting from 1x in PSB1K3
• gingko:

• seq1: es, xp
• da: es, xp
• ra: es, xp
• e0840: ep to get PSB1A3 backbone

• normal:

• seq1: es, ex
• da: es, ex
• ra: es, ex

• ligation:

• 2x seq1, da, ra 2 ways:

• psb1k3
• psb1a3 (gingko)

• transformation onto 14 plates:

• new promoter (g18 on plate 1) in duplicate
• ligation products in duplicate

• planning:

• planned assembly of plasmid construct (cas genes, leader sequence, array) using duet plasmid

Tuesday, July 12

• Transformation results: all successful (14 / 14)

• liquid cultures of:

• promoter
• e0840 (gingko, other)
• 2x seq1, da, ra

• Miniprep using new ethanol protocol:

• 8x (ra, da, seq1)

• nanodropped, yields: 50-1000 ng / ul (hooray)

• psb1a3 red colony

• sequencing orders:

• 1x, 2x, 4x (3 times each)

• submitted primers / synthesis order for our final design:

• new cmr
• new cas3
• new casabcde
• adapter plasmids for leader sequences for cmr, cas
• ordered new restriction enzymes