September/4 September 2009

From 2009.igem.org

1.Colony check

sample No.  (no. of colonies)   
  No.5               0  
  No.9              100~
  No.18              3
  No.20              0
  No.21              1
  No.22              0
  No.23              0
  No.25              0
  No.26              0

No.9 ,18, 26 colony were streaked on plate for quick ligation check.

2.ligation

No.9-I,V , 18-I,V , 24-I,V , 27-I,V

gel cut
↓
purification
↓
ligation 2hr

3.transformation
Transform No.20, 22, 23, 25, 9, 18, 24, 27 and incubate at 37℃ overnight.

4.mini prep Mini prep and nano drop check of above DNA.(1-14L 1-15L 2-14L 1-16I 1-1K 1-15J 1-14N No.21)

sample No.      conc.
  1-14L       14.3 ng/uL
  1-15L        7.1 ng/ul
  2-14L        9.2 ng/uL
  1-16I       11.5 ng/uL
  1-1K        11.1 ng/uL
  1-15J        7.6 ng/uL
  1-14N       15.4 ng/uL
  No.21       19.9 ng/uL

5.digestion and ligation digestion with restriction enzyme

K204005

Vector		Insert
1-18L	8	1-23J	12
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	8	dH₂O	4
total	20uL	total	20uL

↓
37°C , 2hr
↓
gel electrophoresis

No.5-I,V

gel cut
purification
ligation O/N

6.Make LB with agar and Amp plates.

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