August/13 August 2009

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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)
                       1-2M                       100~    (Amp)
                       2-24G                      1?
                       1-12C                      10
                       1-6I                       10<
                       2-8I                       10<
                       2-6E                       10<
                       1-16G                      10
                       1-12A                      10<    
                       2-8O                       10< 
                       
                       2-11N                       10    (Kan)
                       1-18L                       50   

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

after  ligation
              EpsE + terminator                    3        →  liquid medium
             RBS   +  LasR(2-8M)                 10     → rapid check

2.digestion and ligation

digestion with restriction enzyme

K204003

Vector		Insert
(RBS)1-2M	10	cinR(1-14F)	6
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2 Buffer	2	No.2	2
dH₂O	6	dH₂O	10
total	20uL	total	20uL

K204004

Vector		Insert
RBS(1-2M)	10	LasR(2-8M)	10
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	4	dH₂O	6
total	20uL	total	20uL

K204005

Vector		Insert
1-18L	8	1-23J	8
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	8	dH₂O	8
total	20uL	total	20uL

K204006

Vector		Insert
RBS(1-2M)	10	1-14D	10
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	6	dH₂O	8
total	20uL	total	20uL

↓
37°C, 2hr

↓
gel electrophoresis

No. 3 , 4 , 5 , 6 , ladder

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

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

3.ligation check

3.1. EpsE + terminater

Result of Nanodrop concentration check

(sample No.) (concentration)
     2-A               21.5 ng/uL
     2-B               15.8 ng/uL
     2-C               16.7 ng/uL

↓
digestion with restriction enzyme

2-A/2-B/2-C	8
XbaI	1
PstI	1
No.2	2
dH₂O	8
total	20uL

1-23L(vector)	8
XbaI	1
No.2	2
dH₂O	9
total	20uL

↓
37°C over night

3.2. rapid check

(RBS   +  LasR(2-8M))
No.3 and No.8 →  to iquid medium(5mL + Amp 5uL)