August/11 August 2009

From 2009.igem.org

(Difference between revisions)
 
Line 3: Line 3:
  (plate number)-(location on plate) (no. of colonies)
  (plate number)-(location on plate) (no. of colonies)
-
               1-23L                    100>
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]                     100>
-
               1-15N                      10
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N]                     10
-
               2-6P                      10<
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P]                       10<
-
               1-6I                      10<
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I]                       10<
-
               1-12H                      10
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H]                     10
-
               1-18C                      10<
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C]                     10<
-
               1-20F                      ×
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F]                       ×
   
   
  inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
  inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
Line 67: Line 67:
to get new plasmid
to get new plasmid
  each 4uL(DNA) , 1-14F : 2uL
  each 4uL(DNA) , 1-14F : 2uL
-
  1-14H kan
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
-
  1-14F kan
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan
-
  2-18F Amp ('8/10competent cell
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell
   
   
-
  1-23J Amp
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
-
  1-12C Amp (super competent cell??
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell??
 +
 
 +
 
 +
[https://2009.igem.org/Team:Osaka/NOTES back to NOTES]

Latest revision as of 07:46, 12 October 2009

1.before 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)
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]                     100>
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N]                      10
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P]                       10<
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I]                       10<
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H]                      10
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C]                      10<
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F]                       ×

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


2.digestion and ligation

digestion with restriction enzyme

K204001

VectorInsert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M)12[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M)5
SpeI1XbaI1
PstI1PstI1
No.2 Buffer2No.22
dH2O4dH2O11
total20uLtotal20uL


K204002

VectorInsert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)12[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)5
EcoRI1EcoRI1
XbaI1SpeI1
No.22No.22
dH2O4dH2O11
total20uLtotal20uL


37°C , 2hr


gel electrophoresis
Osaka090811.jpg

No.1 No.2 ladder

gel cut

purification by [QIAquick Nucleotide Removal Kit]

ligation

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16°C,overnight


3.Transformation
to get new plasmid

each 4uL(DNA) , 1-14F : 2uL
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell

[http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell??


back to NOTES