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Revision as of 01:11, 10 August 2016
JOURNAL
Projects
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 EBL2 in pSB1C3 construct
Jun
27
Inoculation of strains obtained from Dr. Christopher Hayes
Inoculated overnight cultures of:
- EPI100 w/ EC93 cosmid
- X90 w/ EC869 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)
- 0.75 uL F primer
- 0.75 uL R primer
- 12.5 uL KAPA Hifi 2X MM
- dH2O up to 25 uL
- 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)
- 98C -- 30s
- 98C -- 10s
- TmC -- 30s
- 72C -- ext time
- 72C -- 2min
25uL rxn:
Fragments:
Cycling conditions:
28 cycles
Week
1
Title For The Week
Description blah blah blah
Jun
24
Title for the day (e.g. "Cloning Mutants")
Today we did cloning. fuck cloning. blah blah blah.
