August/11 August 2009

From 2009.igem.org

(Difference between revisions)

Revision as of 08:44, 21 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)
             1-23L                     100>
             1-15N                      10
             2-6P                       10<
             1-6I                       10<
             1-12H                      10
             1-18C                      10<
             1-20F                       ×

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

2.digestion and ligation

digestion with restriction enzyme

K204001

Vector		Insert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M)	12	[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M)	5
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2 Buffer	2	No.2	2
dH2O	4	dH2O	11
total	20uL	total	20uL

K204002

Vector		Insert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)	12	[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)	5
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH2O	4	dH2O	11
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.Transformation
to get new plasmid

each 4uL(DNA) , 1-14F : 2uL
1-14H kan
1-14F kan
2-18F Amp ('8/10competent cell

1-23J Amp
1-12C Amp (super competent cell??

@@ Line 21: / Line 21: @@
 <table border="1" Frame="box">
 <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
-<tr><td>1-2M</td><td>12</td><td>2-8M</td><td>5</td></tr>
+<tr><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M)</td><td>12</td><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M)</td><td>5</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>
@@ Line 35: / Line 35: @@
 <table border="1" Frame="box">
 <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
-<tr><td>1-23L</td><td>12</td><td>3-18O</td><td>5</td></tr>
+<tr><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)</td><td>12</td><td>[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)</td><td>5</td></tr>
 <tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr>
 <tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr>