August/9 October 2009

From 2009.igem.org

We collected sequence data from the automated sequencer and used sequence alignment software to check the sequences against those obtained from the Parts Registry. The results were as follows:

Senders:
LuxI - mismatched
LasI - OK
RhlI - OK
CinI - unconfirmed (DNA sample concentration too low?)

Receivers:
LuxR - OK
LasR - mismatched
RhlR - OK
CinR - unconfirmed (DNA sample concentration too low?)

The DNA for Cin signaling system will be transformed, amplified and purified to obtain at higher concentration and PCR sequencing repeated on them. As time is running out, for the final circuit we shall use parts sent from Chiba University to replace our faulty ones.

2.colony check

 sample   no . of colony
   32                 +++
   X4                  ~30
   X4                    7 
   70                     0
   71                     4
   74                      5
   75                      7

3.min prep

sample   conc.
  1-15J    98 ng/uL 
  1-15L    87
  43         169
  44          172
  52          114
  65         214
  67         148
  67         126

4.transfromation

saple
59 , 72 , 68 , 69 
chiba team parts ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9)

5.digation

K204058

Vector		Insert
1-8E	4	1-8I	10
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	12	dH₂O	6
total	20uL	total	20uL

K204062

Vector		Insert
1-8E	9	33	1
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	7	dH₂O	15
total	20uL	total	20uL

K204072

Vector		Insert
1-1D	5	64	2
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	9	dH₂O	12
total	20uL	total	20uL

K204077

Vector		Insert
1-6I	7	64	2
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	7	dH₂O	12
total	20uL	total	20uL

K204078

Vector		Insert
1-2M	6	1-15N	12
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	10	dH₂O	4
total	20uL	total	20uL

↓
37 degree , 2hr
gel cut & purification
ligation

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