August/11 August 2009

From 2009.igem.org

(Difference between revisions)
Line 1: Line 1:
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コロニー数カウント(8/10にトラフォ行ったもの)
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1.<B>Min prep</b><br>
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  No.   コロニー数
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Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.
 +
 
 +
  (plate number)-(location on plate) (no. of colonies)
  1-23L    100>
  1-23L    100>
  1-15N    10
  1-15N    10
Line 8: Line 10:
  1-18C    10<
  1-18C    10<
  1-20F    ×
  1-20F    ×
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  液培(5mL)にAmp 5uL , Kan 25uL入れて植菌
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 +
inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
 +
 +
2.<B>digestion and ligation</b><br>
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制限酵素処理
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digestion with restriction enzyme
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  Vector
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  Vector1
  1-2M  12
  1-2M  12
  SpeⅠ 1
  SpeⅠ 1
Line 20: Line 25:
  total 20uL
  total 20uL
   
   
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  Insert
+
  Insert1
  2-8M  5
  2-8M  5
  XbaⅠ  1
  XbaⅠ  1
Line 29: Line 34:
   
   
   
   
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  Vector
+
  Vector2
  1-23L  12
  1-23L  12
  EcoRⅠ 1
  EcoRⅠ 1
Line 37: Line 42:
  total  20uL
  total  20uL
   
   
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  Insert
+
  Insert2
  3-18O  5
  3-18O  5
  EcoⅠ  1
  EcoⅠ  1
Line 45: Line 50:
  total  20uL
  total  20uL
   
   
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  2時間ほど37℃で反応させた
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  ↓
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37℃ , 2hr
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↓<br>
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電気泳動
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gel electrophoresis<br>
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ゲル切り出し
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gel cut<br>
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精製を経て
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↓<br>
-
 
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purification by [QIAquick Nucleotide Removal Kit]<br>
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ライゲーション
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↓<br>
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  それぞれ
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ligation
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  DNA溶液    44
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  ligation
 +
  DNA        44
  10* buffer 5
  10* buffer 5
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  ライゲース 1
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  ligation  1
  total      50uL
  total      50uL
 +
↓
 +
16℃ overnight
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トランスフォーメーション
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3.<B>Transformation</b><br>
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  各2uL , 1-14F は 4uL
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to get new plasmid
 +
  each 4uL(DNA) , 1-14F : 2uL
  1-14H kan
  1-14H kan
  1-14F kan
  1-14F kan
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  2-18F Amp ('8/10コンピ
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  2-18F Amp ('8/10competent cell
   
   
  1-23J Amp
  1-23J Amp
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  1-12C Amp (スーパーコンピ
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  1-12C Amp (super competent cell??
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+
-
KanのものはLB培地を加えた後、小さな試験管に入れて振とう培養(1~2hr)してからプレートにまいた
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Revision as of 10:24, 11 August 2009

1.Min prep
Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies. 

(plate number)-(location on plate) (no. of colonies)
1-23L    100>
1-15N    10
2-6P     10<
1-6I     10<
1-12H    10
1-18C    10<
1-20F    ×

inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)


2.digestion and ligation

digestion with restriction enzyme 

Vector1
1-2M  12
SpeⅠ 1
PstⅠ 1
No.2 Buffer 2
dH2O 4
total 20uL

Insert1
2-8M   5
XbaⅠ  1
PstⅠ  1
No.2   2
dH2O   11
total  20 uL


Vector2
1-23L  12
EcoRⅠ 1
XbaⅠ  1
No.2   2
dH2O   4
total  20uL

Insert2
3-18O  5
EcoⅠ  1
SpeⅠ  1
No.2   2
dH2O   11
total  20uL

↓
37℃ , 2hr

↓
gel electrophoresis
gel cut
↓
purification by [QIAquick Nucleotide Removal Kit]
↓
ligation 

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16℃ overnight


3.Transformation
to get new plasmid 

each 4uL(DNA) , 1-14F : 2uL
1-14H kan
1-14F kan
2-18F Amp ('8/10competent cell

1-23J Amp
1-12C Amp (super competent cell??
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