August/13 August 2009

From 2009.igem.org

(Difference between revisions)

Revision as of 08:26, 14 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
Vector1
RBS (1-2M)  10
SpeⅠ 1
PstⅠ 1
No.2 Buffer 2
dH2O 6
total 20uL

Insert1
cinR(1-14F)   6
XbaⅠ  1
PstⅠ  1
No.2   2
dH2O   10
total  20 uL


K204004
Vector2
RBS (1-2M)  10
EcoRⅠ 1
XbaⅠ  1
No.2   2
dH2O   4
total  20uL

Insert2
lasR (2-8M) 10
EcoⅠ  1
SpeⅠ  1
No.2   2
dH2O   6
total  20uL

 
K204005
Vector3
(1-18L)  8
SpeⅠ 1
PstⅠ 1
No.2 Buffer 2
dH2O 8
total 20uL

Insert3
(1-23J)   8
XbaⅠ  1
PstⅠ  1
No.2   2
dH2O   8
total  20 uL
 

 
K204006
Vector4
RBS (1-2M)  10
SpeⅠ 1
PstⅠ 1
No.2 Buffer 2
dH2O 6
total 20uL

Insert4
(1-14D)   10
XbaⅠ  1
PstⅠ  1
No.2   2
dH2O   8
total  20 uL
 

↓
37℃ , 2hr

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

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16℃ 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   8
XbaⅠ  1
PstⅠ  1
No.2   2
dH2O   8
total  20 uL

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

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

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

↓
37℃ over night

3.2. r apid check

No.3 and No.8 →  to iquid medium(5mL + Amp 5uL)

@@ Line 164: / Line 164: @@
 ↓<br>
-℃ over night
+℃ over night<br>
 .2. r apid check
   No.3 and No.8 →  to iquid medium(5mL + Amp 5uL)