Team:HokkaidoU Japan/Notebook/August10

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Single Colony Isolation

  • Observed if any colonies were made
    • Could not see 3-1E
  • Number of other colonies on other plates was also very small
    • Mistake in protocol is inferred
    • Precipitation of cells maybe at fault
  • All but 3-1E was moved to new plates

シングルコロニーアイソレーション

コロニーを確認したところ,3-1Eにはコロニーが見られなかった
ほかのプレートもコロニー数が少なかったため,手順に不完全なところがあったのかもしれない

  • 実験中に沈殿が見られたため
    • コンピテントセルの溶解が不十分だった?
    • 混ぜ方が不十分だった?
  • 2回目の実験で,若干急いだ感じだったので,吸着などが不十分だった?

3-1E以外を新しい培地に移した

PCR Reaction System

Reaction system was prepared for 3 parts, 245 uL in total

Reagent Amount
Autoclaved DW 165 uL
10x PCR buffer 25 uL
2 mM dNTPs 25 uL
25 mM MgSO4 15 uL
EX-F primer 5 uL
PS-R primer 5 uL
KOD plus Neo 5 uL
Total 245 uL
  • 49 uL of reaction solution was added to each of 4 tubes and after DNA template was added
  • Length of each template is listed int the table below
Tube Biobrick Length
1 2-24G(sender) 847 bp
2 1-2M(RBS) 61 bp
3 1-23L(terminator) 178 bp
4 3-1E(heat sensor) 984 bp
  • Template DNA length is calculated by adding prefix and suffix length
    • Biobrick Length + Prefix length + Suffix Length
  • Because Mini prep of 3-1E failed, it amplified by PCR

PCR反応液の調整

== 今回は4パーツ増幅のためTotal 245 uL作成した.

Autoclaved DW 165 uL
10x PCR buffer 25 uL
2 mM dNTPs 25 uL
25 mM MgSO4 15 uL
EX-F primer 5 uL
PS-R primer 5 uL
KOD plus Neo 5 uL
Total 245 uL
  • 反応液49 uLを4本のPCRチューブに分注し,Templateを1 uLずつ加えた
  • それぞれのPCRチューブとTemplate,lengthは
1 2-24G(sender) 847 bp
2 1-2M(RBS) 61 bp
3 1-23L(terminator) 178 bp
4 3-1E(heat sensor) 984 bp
  • DNA lengthはパーツ長+プライマー(49 bp)
  • 3-1EはTransformationがうまくいかなかったため

PCR program

Predenature 94℃ 2 min
Denature 98℃ 10 sec
Extension 68℃ 30 sec
Hold 4℃
  • 30 cycles
  • KOD potency is 30 sec/kbp, so 30 sec for1cycle

Electrophoresis

Electrophoresis of amplified BioBricks
  • Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
  • Used marker pUC1d19/Hinf1
  • Added 20 uL of EtBr to 1/2 TBE
Lane DNA Length of DNA
1 pUC1d19/Hinf1
2 2-24G(sender) 847 bp
3 1-2M(RBS) 61 bp
4 1-23L(terminator) 178 bp
5 3-1E(heat sensor) 984 bp

電気泳動

  • 増幅したDNAを5 uLとって6xサンプルバッファー1 uLを加えた
  • マーカーはpUC119/Hinf1を使用
  • エチブロは20 uL使用
short discription