August/11 August 2009

From 2009.igem.org

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

Vector		Insert
[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
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2 Buffer	2	No.2	2
dH₂O	4	dH₂O	11
total	20uL	total	20uL

K204002

Vector		Insert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)	12	[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)	5
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	4	dH₂O	11
total	20uL	total	20uL

↓
37°C , 2hr

↓
gel electrophoresis

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??

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