Team:HokkaidoU Japan/Notebook/October2

From 2010.igem.org

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==3 Piece Ligation: Retry==
==3 Piece Ligation: Retry==
===[[Team:HokkaidoU_Japan/Protocols|Gel Extraction]]===
===[[Team:HokkaidoU_Japan/Protocols|Gel Extraction]]===

Revision as of 11:39, 16 October 2010

Notebook
Fig.1

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

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

2 piece ligation of T3SS signal and pSB1C3 for DNA Submission

Fig.2

T3SS signal

Reagent Amount
10x M buffer 2 uL
DW 12
DNA 3
BSA 2
EcoR I 0.5
Pst I 0.5
Total 20 uL

pSB1C3

Reagent Amount
10x M buffer 2 uL
DW 13
DNA 2
BSA 2
EcoR I 0.5
Pst I 0.5
Total 20 uL

->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))
Fig.3

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
Fig.4
Fig.5

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

Fig.6
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)
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