Transformation Results

Colony Check

Plate	Colonies
M 34	no
M 12	yes
I 34	no
I 34	yes
1-3A 34	yes
1-3A 12	yes

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• 1-3A had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
  • So the medium and antibiotic consentration seems ok,
• Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
• Colonies of 1-3A and 12 were obviously different

→ So we suspected issues in Ligation and DNA concentration bused for transformation

Estimation of 1-3A Consentration

• Added 1 uL of 1-3A into 30 uL of competent cells
• Transformation was successful
• Electrophoresed 1 uL of 1-3A，calculated amount of transformed DNA

→Estimated concentration 2 ng/uL

Ethanol precipitation of pSB1C3 and Ligation

Ethanol precipitation

1. Added 8.2 uL of sodium acetate [3 M]
2. Added 205 uL of Ethanol [100%] and mixed
3. Centrifuge at 15,00rpm, 4C for 10 min
4. Discarded supernatant and added 500 uL of Ethanol [70%], mixed
5. Centrifuge at 15,00rpm, 4C for 5 min
6. Discarded supernatant
7. Dried via vacuum desiccator
8. Added 8.2 uL of ADW を加え，元の10倍に濃縮する

• Concentration increased 10 fold

Ligation

1. Mixed 8.2 uL of DNA solution with the same volume of Ligation Solution
2. Added extra 1 uL of T4 ligase
3. Incubated at 16C for 30 min

Tomorrow will do aditional Electrophoresis gel extraction and transformation

らおりプライマーで作ったPCR productsのDigestion

前日のPCRから，増やすことのできたパーツのDNA量を求めた

RBS	27 ng/uL	312 bp
GFP	54 ng/uL	1020 bp
dT	49 ng/uL	429 bp

Digestion 反応系

Reagent	Amount
10x M buffer	1 uL
DW	4.5
DNA	2.5
BSA	1
EcoR I	0.5
Pst I	0.5
Total	10 uL

→37℃で60分

→ラオリプライマーの真価を電気泳動で確認