August/13 August 2009

From 2009.igem.org

(Difference between revisions)

Revision as of 12:57, 16 August 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)
                        1-2M                      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
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)

@@ Line 32: / Line 32: @@
 <table border="1" Frame="box">
 <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
-<tr><td>(RBS)1-2M</td><td>10</td><td>cin(1-14F)</td><td>6</td></tr>
+<tr><td>(RBS)1-2M</td><td>10</td><td>cinR(1-14F)</td><td>6</td></tr>
 <tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr>
 <tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr>