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)
             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

Vector		Insert
RBS (1-2M)	12	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
terminator (1-23L)	12	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
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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