Team:HokkaidoU Japan/Notebook/October2

Colony PCR

• Performed Colonie PCR for 3 colonies which were incubated over night (Fig.1)
• Colony numbers were: 1, 2 and 3
• Results showed no insertion
  • but when result came, had already done miniprep and prepared Sequencing Master Mix

Miniprep

• Used Qiagen kit
• Melted in 50 uL TE instead of H₂O

Preparation for Sequencing

• Mixed as shown in the table below

5x Sequencing Buffer 1.5uL 24.75 uL Ready Reaction Premix 1 uL 16.5 H2O 5 uL 80 uL total 7.5/sample

Reagent	Amount	Amount for 16.5
5x Sequencing Buffer	1.5uL	24.75 uL
Ready Reaction Premix	1 uL	16.5
DW	5 uL	80 uL
Total	7.5/sample	121.25

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3 Piece Ligation: Retry

Gel Extraction

Gel Extraction of DNAs that had been cut on October 1

• Couldn't see T3SS signal's bands, retried digestion like below

Digestion: Retry

Reagent	Amount
10x M buffer	10 uL
DW	64
T3SS signal	6
BSA	10
Xba I	9.6
Pst I	0.4
Total	100 uL

-> Electrophoresed with other samples

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2 piece ligation of T3SS signal and pSB1C3 for DNA Submission

->Incubated at 37C for 2 hrs

• Electrophoresis (Fig.2: λ/HindIII 2uL, T3SS signal, pSB1C3, T3SS signal for 3 piece ligation(applied to 2 wells))

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Colony PCR (previous day's 3 piece ligation)

Did colony PCR of latecomers (No.5 to No.16)(Fig.3)

• No.8, 10, 11, 12, 15 looked like good
• Transfered No.8, 10, 11, 12, 13, 14, 15, 16(control) to 2 mL LBT (Tetracycline 15 ug/mL) and started incubation

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Colony PCR (T3SS signal)

Did colony PCR to amplify T3SS signal (from BAC clone that has T3SS signal insertion)

• Colony solution 7 uL, quick Taq 10 uL, primer(Forward and Reverse) 1.5 uL each
• Extention: 90 sec, 40 cycles

Electrophoresed to purify the DNA via gel extraction (Fig.4)
Electrophoresed to estimate the concentration of the DNA (Fig.5: λ/HindIII 2 uL, DNA solution 1 uL)

• Concentration was estimated at 40 ng/uL

Digestion

Reagent	Amount
10x H buffer	2 uL
DW	9
DNA	5
BSA	2
EcoR I	1
Pst I	1
Total	20 uL

->Electrophoresis, Gel Extraction (Fig.6)