August/11 August 2009

From 2009.igem.org

(Difference between revisions)
Line 48: Line 48:
↓<br>
↓<br>
gel electrophoresis<br>
gel electrophoresis<br>
 +
[[Image:Osaka090811.jpg]]<br>
 +
No.1 No.2 ladder
gel cut<br>
gel cut<br>
↓<br>
↓<br>

Revision as of 08:56, 26 September 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)
             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

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
1-14H kan
1-14F kan
2-18F Amp ('8/10competent cell

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