Team:Chiba/Notebook 2

From 2010.igem.org

(Difference between revisions)
Line 197: Line 197:
   Rev : pSB1C3 FASTR-R<br>
   Rev : pSB1C3 FASTR-R<br>
   -------------------------
   -------------------------
-
   template      5㎕
+
   template      5uL
-
   primer Fwd    5㎕
+
   primer Fwd    5uL
-
   primer Rev    5㎕
+
   primer Rev    5uL
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer      5㎕
+
   10xbuffer      5uL
-
   NFW            24㎕
+
   NFW            24uL
-
   VentP          1㎕
+
   VentP          1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  PCR(TAKARA)
  PCR(TAKARA)
   94°C – 5min
   94°C – 5min
Line 220: Line 220:
   4. control<br>
   4. control<br>
   -------------------------
   -------------------------
-
   template      3㎕
+
   template      3uL
-
   primer Fwd    5㎕
+
   primer Fwd    5uL
-
   primer Rev    5㎕
+
   primer Rev    5uL
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer    5㎕
+
   10xbuffer    5uL
-
   NFW          26㎕
+
   NFW          26uL
-
   VentP        1㎕
+
   VentP        1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  Using primer
  Using primer
   Fwd : pSB1C3 FASTR-F
   Fwd : pSB1C3 FASTR-F
Line 247: Line 247:
FASTR Cloning (Plasmid1)
FASTR Cloning (Plasmid1)
  Vector (about 2kbp)
  Vector (about 2kbp)
-
  -pSB1A3 (conservative solution 100ng/㎕)<br>
+
  -pSB1A3 (conservative solution 100ng/uL)<br>
  Insert (about 1kbp)
  Insert (about 1kbp)
-
   1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
   1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
-
   2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
   2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
-
   3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
   3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
-
   4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)<br>
+
   4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)<br>
  master mix
  master mix
  ---------------------------------------------------------------------
  ---------------------------------------------------------------------
Line 265: Line 265:
  NFW                   3 12
  NFW                   3 12
  -----------------------------------------------------------------------
  -----------------------------------------------------------------------
-
  Total          10㎕ 28㎕
+
  Total          10uL 28uL
  Room temperature 2h
  Room temperature 2h
Transformation
Transformation
-
  Using cell strain : BL21(DE3) 50㎕
+
  Using cell strain : BL21(DE3) 50uL
  Using plasmid
  Using plasmid
-
   L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
+
   L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5uL
-
   L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
+
   L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5uL
-
   L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
+
   L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL
-
   L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
+
   L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL
After transformation
After transformation
  We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates.
  We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates.
Line 284: Line 284:
<html><h3>2010-09-23</h3></html>
<html><h3>2010-09-23</h3></html>
From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3.
From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3.
-
And then it is eluted by 50㎕ Elution Solution.
+
And then it is eluted by 50uL Elution Solution.
↓
↓
-
Gel Electrophoresis(1㎕)
+
Gel Electrophoresis(1uL)
-
  L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
+
  L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5uL
-
  L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
+
  L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5uL
-
  L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
+
  L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL
-
  L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
+
  L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL
  Remaining of above sample is transformed and incubated.
  Remaining of above sample is transformed and incubated.
-
  cell strain : XL10G(50㎕/tube)
+
  cell strain : XL10G(50uL/tube)
  plasmid : L1~L4(total 4)
  plasmid : L1~L4(total 4)
FASTR Cloning (Plasmid1)
FASTR Cloning (Plasmid1)
  Vector (about 2kbp)
  Vector (about 2kbp)
-
   -pSB1A3 (conservative solution 100ng/㎕)
+
   -pSB1A3 (conservative solution 100ng/uL)
  Insert (about 1kbp)
  Insert (about 1kbp)
-
   1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
   1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
-
   2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
   2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
-
   3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
   3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
-
   4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
   4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
  master mix
  master mix
  ---------------------------------------------------------------------
  ---------------------------------------------------------------------
Line 314: Line 314:
  NFW                   3 12
  NFW                   3 12
  -----------------------------------------------------------------------
  -----------------------------------------------------------------------
-
  Total               10㎕ 28㎕
+
  Total               10uL 28uL
  Room temperature 2h
  Room temperature 2h
After ligation plasmid is transformed and incubated in LB broth.
After ligation plasmid is transformed and incubated in LB broth.
-
cell strain : BL21(DE3), XL10G(each 50㎕/tube)
+
cell strain : BL21(DE3), XL10G(each 50uL/tube)
plasmid : L1~L4
plasmid : L1~L4
   
   
Line 323: Line 323:
At 23th September
At 23th September
Remaining of above sample is transformed and incubated.
Remaining of above sample is transformed and incubated.
-
cell strain : XL10G(50㎕/tube)
+
cell strain : XL10G(50uL/tube)
plasmid : L1~L4(total 4)
plasmid : L1~L4(total 4)
We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.<br>
We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.<br>
Line 330: Line 330:
  --------------------------------------------------------------------------
  --------------------------------------------------------------------------
  Template(picked colonies)
  Template(picked colonies)
-
  Primer VF         2㎕ 40㎕
+
  Primer VF         2uL 40uL
-
  Primer VR         2㎕ 40㎕
+
  Primer VR         2uL 40uL
-
  10x Buffer         2㎕ 40㎕
+
  10x Buffer         2uL 40uL
-
  dNTP                 2㎕ 40㎕
+
  dNTP                 2uL 40uL
-
  NFW                 10㎕ 200㎕
+
  NFW                 10uL 200uL
-
  Taq DNA Polymerase 0.5㎕ 5㎕
+
  Taq DNA Polymerase 0.5uL 5uL
  --------------------------------------------------------------------------
  --------------------------------------------------------------------------
-
  Total                 20㎕ 400㎕
+
  Total                 20uL 400uL
  ↓
  ↓
  PCR
  PCR
Line 366: Line 366:
  (Digestion)
  (Digestion)
             Insert(J01011)   Vector(pSB3C5)
             Insert(J01011)   Vector(pSB3C5)
-
  DNA       10㎕                 10㎕
+
  DNA       10uL                 10uL
-
  NFW       32.5㎕               32.5㎕
+
  NFW       32.5uL               32.5uL
-
  NEB Buffer 2 5㎕                 5㎕
+
  NEB Buffer 2 5uL                 5uL
-
  BSA       0.5㎕               0.5㎕
+
  BSA       0.5uL               0.5uL
-
  EcoRI         1㎕               1㎕
+
  EcoRI         1uL               1uL
-
  PstI         1㎕               1㎕
+
  PstI         1uL               1uL
  ------------------------------------------------------------------------
  ------------------------------------------------------------------------
-
  total                      50㎕               50㎕
+
  total                      50uL               50uL
  ↓37°C 15min incubator
  ↓37°C 15min incubator
  ↓65°C 20min (sterilization dryer)
  ↓65°C 20min (sterilization dryer)
  ※After digestion, we did gel electrophoresis<br>
  ※After digestion, we did gel electrophoresis<br>
  (Ligation)
  (Ligation)
-
   13㎕ NFW
+
   13uL NFW
-
     2㎕ Insert(digested)
+
     2uL Insert(digested)
-
     2㎕ Vector(digested)                  total 20㎕ in 0.2mL PCR tube
+
     2uL Vector(digested)                  total 20uL in 0.2mL PCR tube
-
     2㎕ 10x T4 DNA Ligase Buffer
+
     2uL 10x T4 DNA Ligase Buffer
-
     1㎕ T4 DNA Ligase<br>
+
     1uL T4 DNA Ligase<br>
  ↓10min at room temperature
  ↓10min at room temperature
  ↓20min at 65°C (sterilization dryer)<br>
  ↓20min at 65°C (sterilization dryer)<br>
  (Transformation)
  (Transformation)
-
  Transformation XL10G 50㎕ to 10㎕ of ligation product
+
  Transformation XL10G 50uL to 10uL of ligation product
  ↓37°C
  ↓37°C
  Insert check of living colonies(colony PCR)    /    liquid incubation
  Insert check of living colonies(colony PCR)    /    liquid incubation
Line 393: Line 393:
  --------------------------------------------
  --------------------------------------------
  Template(picked colonies)                                  ↓
  Template(picked colonies)                                  ↓
-
  Primer VF 2㎕                                     Gel electrophoresis
+
  Primer VF 2uL                                     Gel electrophoresis
-
  Primer VR 2㎕
+
  Primer VR 2uL
-
  10x Buffer 2㎕
+
  10x Buffer 2uL
-
  dNTP 2㎕
+
  dNTP 2uL
-
  NFW 11.5㎕
+
  NFW 11.5uL
-
  Taq DNA Polymerase 0.5㎕
+
  Taq DNA Polymerase 0.5uL
  --------------------------------------------------------
  --------------------------------------------------------
-
  Total 20㎕
+
  Total 20uL
  ↓
  ↓
  PCR
  PCR

Revision as of 15:57, 26 October 2010




 




2010-09-21

PCR 

Because it didn’t come out vector PCR(gel electrophoresis), 
we operated re-experiment to change template and template amounts.

Using template
 1. Biobrick 2010 1-3A(pSB1C3)
 2. Biobrick 2010 pSB1C3
 3. Biobrick 2010 2-9A(pSB1A3)
 4. control

Using primer
 Fwd : pSB1C3 FASTR-F
 Rev : pSB1C3 FASTR-R

 -------------------------
 template       5uL
 primer Fwd     5uL
 primer Rev     5uL
 dNTP           5uL
 10xbuffer      5uL
 NFW            24uL
 VentP          1uL
 -------------------------
                50uL

PCR(TAKARA)
 94°C – 5min
 94°C – 15sec    --
 51°C – 30sec      │- 25 cycles
 72°C – 1min     -- 
 72°C – 10min

Vector PCR 

Using template
 1. Biobrick 2010 1-3A(pSB1C3)
 2. Biobrick 2010 pSB1C3
 3. Biobrick 2010 2-9A(pSB1A3)
 4. control

 -------------------------
 template      3uL
 primer Fwd    5uL
 primer Rev    5uL
 dNTP          5uL
 10xbuffer     5uL
 NFW           26uL
 VentP         1uL
 -------------------------
               50uL

Using primer
 Fwd : pSB1C3 FASTR-F
 Rev : pSB1C3 FASTR-R

PCR Condition
 94°C – 5min
 94°C – 30sec      --
 51°C – 30sec        │-25 cycles
 72°C – 2min       --
 72°C – 10min

2010-09-22

Biobrick 2010 2-9A(pSB1A3) -> Gel extraction Gel Electrophoresis 

Biobrick 2010 2-9A(pSB1A3), Biobrick 2010 1-3A(pSB1C3), Biobrick 2010 pSB1C3, Biobrick 2010 2-9A(pSB1A3)
As a result, it didn’t come out band of 1-3A and pSB1C3. 
We concluded re-experiment from liquid incubation to mini-prep. Must check gel electrophoresis band after mini-prep.

FASTR Cloning (Plasmid1) 

Vector (about 2kbp)
-pSB1A3 (conservative solution 100ng/uL)

Insert (about 1kbp)
 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)

master mix
---------------------------------------------------------------------
Vector(50ng)         	0.5	2
Insert(75ng)	          3     -
Tango buffer	        0.5	2
T4 Ligase buffer	0.5	2
ATP	                  1	4
LguI	                0.5	2
Ligase           	0.5	2
DpnI	                0.5	2
NFW	                  3	12
-----------------------------------------------------------------------
Total           	10uL	28uL
Room temperature 2h

Transformation 

Using cell strain : BL21(DE3) 50uL
Using plasmid
 L1 : Pet-LuxR-PT7/cI(OR2)-GFP   5uL
 L2 : Pet-LuxR-PT7/cI(OR1)-GFP   5uL
 L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL
 L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL

After transformation 

We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates.
※A Reason of using DE3 cell strain
DE3 cell strain has T7 RNA Polymerase gene in genome, and under IPTG derivation T7RNAP is expressed.
Plasmid1 has  T7/cI hybrid promoter. 
So if this promoter is accelerated by T7RNAP, GFP is expressed and shines green. 
In other words, we used DE3 cell strain to check T7 accelerating function of PT7/cI.

2010-09-23

From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3. And then it is eluted by 50uL Elution Solution. ↓ Gel Electrophoresis(1uL) 

L1 : Pet-LuxR-PT7/cI(OR2)-GFP   5uL
L2 : Pet-LuxR-PT7/cI(OR1)-GFP   5uL
L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL
Remaining of above sample is transformed and incubated.
cell strain : XL10G(50uL/tube)
plasmid : L1~L4(total 4)

FASTR Cloning (Plasmid1) 

Vector (about 2kbp)
 -pSB1A3 (conservative solution 100ng/uL)
Insert (about 1kbp)
 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
master mix
---------------------------------------------------------------------
Vector(50ng)         	0.5	2
Insert(75ng)	          3	-
Tango buffer	        0.5	2
T4 Ligase buffer	0.5	2
ATP	                  1	4
LguI	                0.5	2
Ligase	                0.5	2
DpnI	                0.5	2
NFW	                  3	12
-----------------------------------------------------------------------
Total	               10uL	28uL
Room temperature 2h

After ligation plasmid is transformed and incubated in LB broth. cell strain : BL21(DE3), XL10G(each 50uL/tube) plasmid : L1~L4

2010-09-24

At 23th September Remaining of above sample is transformed and incubated. cell strain : XL10G(50uL/tube) plasmid : L1~L4(total 4) We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.
Insert Check Colony PCR(Plasmid 1) 

master mix
--------------------------------------------------------------------------
Template(picked colonies)
Primer VF	         2uL	40uL
Primer VR	         2uL	40uL
10x Buffer	         2uL	40uL
dNTP	                 2uL	40uL
NFW	                10uL	200uL
Taq DNA Polymerase	0.5uL	5uL
--------------------------------------------------------------------------
Total	                20uL	400uL
↓
PCR
 94°C – 5min
 94°C – 30sec      --
 51°C – 30sec        │-20 cycles
 72°C – 1min       --
 72°C – 10min
↓
Gel Electrophoresis

2010-09-25

Mini-prep and gel electrophoresis of liquid culture medium at 24th September. 

Gel Electrophoresis picture
↓
Transformation to BL21(DE3) 
↓
Incubation in LB broth, IPTG(0, 10, 100 μM) 
↓37°C
About total sample, it is shown GFP ON/OFF switching on eyes.
↓
For the purpose of measuring fluorescence level, we did liquid incubation from each plate.
IPTG(0, 10, 100 μM) x 6 samples = 18 test tubes
↓37°C
∙ measuring fluorescence level(liquid incubation)
∙ pelletization of cell

Digestion and ligation of BBa-J01011(Ptet-cI) and pSB3C5 

(Digestion)
           Insert(J01011)	   Vector(pSB3C5)
DNA	       10uL	                10uL
NFW	       32.5uL	               32.5uL
NEB Buffer 2	5uL	                5uL
BSA	       0.5uL	               0.5uL
EcoRI	        1uL	               1uL
PstI	        1uL	               1uL
------------------------------------------------------------------------
total                      50uL                50uL
↓37°C 15min incubator
↓65°C 20min (sterilization dryer)
※After digestion, we did gel electrophoresis

(Ligation)
  13uL NFW
   2uL Insert(digested)
   2uL Vector(digested)                  total 20uL in 0.2mL PCR tube
   2uL 10x T4 DNA Ligase Buffer
   1uL T4 DNA Ligase

↓10min at room temperature
↓20min at 65°C (sterilization dryer)

(Transformation)
Transformation XL10G 50uL to 10uL of ligation product
↓37°C
Insert check of living colonies(colony PCR)     /     liquid incubation
↓total 7 colonies                               ↓37°C
Insert Check Colony PCR(Plasmid 1)	                    ↓We did mini-prep only No.4 sample checked insert.
--------------------------------------------
Template(picked colonies)                                  ↓
Primer VF	2uL	                                    Gel electrophoresis
Primer VR	2uL	
10x Buffer	2uL
dNTP	2uL
NFW	11.5uL
Taq DNA Polymerase	0.5uL
--------------------------------------------------------
Total	20uL
↓
PCR
 94°C 5min
 94°C 30sec   --
 51°C 30sec     │-30 cycle
 72°C 1min    --
 72°C 10min
↓
Gel electrophoresis
Retrieved from "http://2010.igem.org/Team:Chiba/Notebook_2"