Team:UCLA/Notebook

JOURNAL

P R O J E C T S

CDI

Protein Cages

Week

1

Construction of ATCC 13048 Loci 2 in pSB1C3

Isolation of template DNA from EC93, EC869, and ATCC 13048; PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3 construct

Jun

27

Inoculation of strains obtained from Dr. Christopher Hayes

Inoculated overnight cultures of:

  • EPI100 w/ EC869 cosmid
  • X90 w/ EC93 cosmid
  • ATCC 13048

Conditions:

  • 10mL LB
  • 10uL ampicillin
  • 37 C for 16h shaking
Jun

28

Isolation of template DNA

Miniprep of EC93 and EC869

  • EC93 #1: 145.42 ng/μL
  • EC93 #2: 198.85 ng/μL
  • EC869 #1: 90.81 ng/μL
  • EC869 #2: 45.95 ng/μL

gDNA isolation of ATCC 13048

  • 34.87 ng/μL

Gibson primers designed for EBL1 and EBL2 toxin switched constructs in pSB1C3 and pSC101

Jun

29

PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3

50uL rxn:

  • 2.5 uL F primer
  • 2.5 uL R primer
  • 25 uL Q5 2X MM
  • 19 uL dH2O

Fragments:

  • EBL1 in pSB1C3 frag 2
  • EBL2 in pSB1C3 frag 2
  • EBL2 in pSB1C3 frag 3

Cycling conditions:

  • 98C -- 30s
  • 98C -- 10s
  • TmC -- 30s
  • 72C -- ext time
    •
  • 72C -- 2min

28 cycles

Jun

30

Troubleshooting PCR of Gibson fragments
  • try using KAPA Hifi instead of Q5
  • add more template gDNA (1ng, 10ng, 35ng, 70ng)

    • 25uL rxn:

    • 0.75 uL F primer
    • 0.75 uL R primer
    • 12.5 uL KAPA Hifi 2X MM
    • dH2O up to 25 uL

    Fragments:

    • EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 140s)
    • EBL2 in pSB1C3 frag 2 (Tm = 65C, ext time = 88s)
    • EBL2 in pSB1C3 frag 3 (Tm = 65C, ext time = 180s)

    Cycling conditions:

    • 98C -- 30s
    • 98C -- 10s
    • TmC -- 30s
    • 72C -- ext time
      •
    • 72C -- 2min

    28 cycles

    ladder; EBL2 frag 2; EBL1 frag 2; EBL2 frag 3

Week

2

Construction of ATCC 13048 Loci 2 in pSB1C3 Cont.

PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3 construct

Jul

5

PCR of EBL1 and EBL2 Gibson fragments for pSB1C3

    Redoing failed PCR, modified protocol by having elongation time be extended to be 30s/kb; annealing temperature set to 72C; ran 25uL and 50uL rxn

    Fragments:

    • EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 210s)
    • EBL2 in pSB1C3 frag 1 (Tm = 65C, ext time = 180s)
    • EBL2 in pSB1C3 frag 4 (Tm = 64C, ext time = 120s)

    Cycling conditions:

    • 98C -- 30s
    • 98C -- 10s
    • Tm -- 30s
    • 72C -- ext time
      •
    • 72C -- 2min
    • 14C -- infinite

    28 cycles

    ladder; EBL2 frag 1; EBL2 frag 4; EBL1 frag 2 (25uL); EBL1 frag 2 (50uL)

  • only primer dimers observed

Jul

6

Troubleshooting PCR of EBL1 frag 2, EBL2 frag 1, EBL2 frag 4

    Redoing failed PCR, modified protocol by doubling and quadrupling template dna amount; changed from 28 to 32 cycles

  • cycling conditions remained constant

    • ladder; 2x EBL1 frag 2; 4x EBL1 frag 2; 2x EBL1 frag 1; 4 EBL1 frag 1; 2x EBL1 frag 4; 4x EBL1 frag 4

  • doubling template DNA resolved EBL2 frag 2 and 4 problem; most likely faulty primers for EBL1 frag 2

Jul

7

Transformation of DH5-alpha with EC93 cosmid and pSK33
  • transform with 1ng of DNA
  • EC93: AmpR; pSK33: KanR
    • Jul

    8

    Troubleshooting PCR of EBL1 frag 2 in pSB1C3 Cont.
  • redid protocol in 7/6/16 with newly diluted primers

    • inoculated starter cultures of DH5-alpha w/ EC93, DH5-alpha w/ pSK33, and X90 w/ EC93

    Jul

    9

    Verification of EC93 cosmid and miniprepping for more pSK33
  • pSK33 had poor yield since it is a pSC101 derivative and is very low copy --> next time miniprep 10mL of culture

    • ran test digest to confirm EC93 cosmid:

    • 500 ng DNA
    • 5uL NEB 2.1
    • 1uL EcoRI
    • H2O to 50uL

    expecting bands at 10kb and 8kb

    ladder;EC93 cosmid from DH5-alpha;EC93 cosmid from X90;ladder