Team:Chiba/Notebook 1

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Gel Electrophoresis
Gel Electrophoresis
-
 
-
 
-
<html><h3>2010-09-21</h3></html>
 
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PCR
 
-
Because it didn’t come out vector PCR(gel electrophoresis),
 
-
we operated re-experiment to change template and template amounts.<br>
 
-
Using template
 
-
  1. Biobrick 2010 1-3A(pSB1C3)
 
-
  2. Biobrick 2010 pSB1C3
 
-
  3. Biobrick 2010 2-9A(pSB1A3)
 
-
  4. control<br>
 
-
Using primer
 
-
  Fwd : pSB1C3 FASTR-F
 
-
  Rev : pSB1C3 FASTR-R<br>
 
-
  -------------------------
 
-
  template      5㎕
 
-
  primer Fwd    5㎕
 
-
  primer Rev    5㎕
 
-
  dNTP          5㎕
 
-
  10xbuffer      5㎕
 
-
  NFW            24㎕
 
-
  VentP          1㎕
 
-
  -------------------------
 
-
                50㎕<br>
 
-
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<br>
 
-
  -------------------------
 
-
  template      3㎕
 
-
  primer Fwd    5㎕
 
-
  primer Rev    5㎕
 
-
  dNTP          5㎕
 
-
  10xbuffer    5㎕
 
-
  NFW          26㎕
 
-
  VentP        1㎕
 
-
  -------------------------
 
-
                50㎕<br>
 
-
Using primer
 
-
  Fwd : pSB1C3 FASTR-F
 
-
  Rev : pSB1C3 FASTR-R<br>
 
-
PCR Condition
 
-
  94°C – 5min
 
-
  94°C – 30sec      --
 
-
  51°C – 30sec        │-25 cycles
 
-
  72°C – 2min      --
 
-
  72°C – 10min
 
-
 
-
<html><h3>2010-09-22</h3></html>
 
-
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/㎕)<br>
 
-
Insert (about 1kbp)
 
-
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
  4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)<br>
 
-
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          10㎕ 28㎕
 
-
Room temperature 2h
 
-
Transformation
 
-
Using cell strain : BL21(DE3) 50㎕
 
-
Using plasmid
 
-
  L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
 
-
  L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
 
-
  L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
 
-
  L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
 
-
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.
 
-
 
-
<html><h3>2010-09-23</h3></html>
 
-
From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3.
 
-
And then it is eluted by 50㎕ Elution Solution.
 
-
↓
 
-
Gel Electrophoresis(1㎕)
 
-
L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
 
-
L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
 
-
L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
 
-
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
 
-
Remaining of above sample is transformed and incubated.
 
-
cell strain : XL10G(50㎕/tube)
 
-
plasmid : L1~L4(total 4)
 
-
FASTR Cloning (Plasmid1)
 
-
Vector (about 2kbp)
 
-
  -pSB1A3 (conservative solution 100ng/㎕)
 
-
Insert (about 1kbp)
 
-
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
  4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
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               10㎕ 28㎕
 
-
Room temperature 2h
 
-
After ligation plasmid is transformed and incubated in LB broth.
 
-
cell strain : BL21(DE3), XL10G(each 50㎕/tube)
 
-
plasmid : L1~L4
 
-
 
-
<html><h3>2010-09-24</h3></html>
 
-
 
-
At 23th September
 
-
Remaining of above sample is transformed and incubated.
 
-
cell strain : XL10G(50㎕/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 2㎕ 40㎕
 
-
Primer VR 2㎕ 40㎕
 
-
10x Buffer 2㎕ 40㎕
 
-
dNTP 2㎕ 40㎕
 
-
NFW 10㎕ 200㎕
 
-
Taq DNA Polymerase 0.5㎕ 5㎕
 
-
--------------------------------------------------------------------------
 
-
Total 20㎕ 400㎕
 
-
 
-
↓
 
-
PCR
 
-
94°C – 5min
 
-
94°C – 30sec
 
-
51°C – 30sec        20 cycles
 
-
72°C – 1min
 
-
72°C – 10min
 
-
↓
 
-
gel electrophoresis
 
-
 
-
<html><h3>2010-09-25</h3></html>
 
-
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       10㎕                 10㎕
 
-
NFW       32.5㎕               32.5㎕
 
-
NEB Buffer 2 5㎕                 5㎕
 
-
BSA       0.5㎕               0.5㎕
 
-
EcoRI         1㎕               1㎕
 
-
PstI         1㎕               1㎕
 
-
------------------------------------------------------------------------
 
-
total                      50㎕                50㎕
 
-
↓37°C 15min incubator
 
-
↓65°C 20min (sterilization dryer)
 
-
※After digestion, we did gel electrophoresis<br>
 
-
(Ligation)
 
-
  13㎕ NFW
 
-
    2㎕ Insert(digested)
 
-
    2㎕ Vector(digested)                  total 20㎕ in 0.2mL PCR tube
 
-
    2㎕ 10x T4 DNA Ligase Buffer
 
-
    1㎕ T4 DNA Ligase<br>
 
-
↓10min at room temperature
 
-
↓20min at 65°C (sterilization dryer)<br>
 
-
(Transformation)
 
-
Transformation XL10G 50㎕ to 10㎕ 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 2㎕                                     Gel electrophoresis
 
-
Primer VR 2㎕
 
-
10x Buffer 2㎕
 
-
dNTP 2㎕
 
-
NFW 11.5㎕
 
-
Taq DNA Polymerase 0.5㎕
 
-
--------------------------------------------------------
 
-
Total 20㎕
 
-
↓
 
-
PCR
 
-
  94°C 5min
 
-
  94°C 30sec  --
 
-
  51°C 30sec    │-30 cycle
 
-
  72°C 1min    --
 
-
  72°C 10min
 
-
↓
 
-
Gel electrophoresis
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
==Notebook==
==Notebook==

Revision as of 14:32, 21 October 2010




2010-08-31

Making plate with Broth 

Amp x 13 
Kan x 1 
Amp + IPTG x 1

Transformation 

R0010 B0030 B0032 B0033 K145270 I746903 R0051 C0051 K145001 C0012 K145001 C0012 P0312 I719015

2010-09-02

Mini-prep(DNA transformated in August 31th) 

I13522 C0040 C0052 C0053 C0080 C0072 I0500 Q04510 Q04400 Q04121 R0040 B0014
B1101 I715038 K081012 K082034 K113007 I763011

2010-09-03

Preparing for experiment 

We inserted plasmid with cI promoter + GFP to E.coli. And

2010-09-04

Preparing for experiment 

I746903

2010-09-05

K091107 R0065 K145150 Q01121 Q01511 Q03121 Q03400 Q03530 Q04511 J06800 J06801

2010-09-06

Co-transformation 

Plux – gfp + Pc each 1㎕
SOC 200㎕

2010-09-07

E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121

2010-09-08

Transformation 

XL10GkanR 30㎕ DNA 1㎕ SOC 100㎕
K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034

2010-09-14

1) K091204(2-8J) Pc – luxR function check
  [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1㎕ + XL10GkanR 50㎕
  AHL : 30C8HSL(1/10000 of 1μM)
2) T9002

2010-09-16

 PCR
  T7/cI(OR1/2) – GFP 
              (1)      (2)
  --------------------------------
   template   1㎕
   primer     5㎕
   F          5㎕
   R          5㎕
   10x buffer 5㎕
   dNTP       5㎕
   NFW        28㎕
   ventP      1㎕
  -------------------------------
             50㎕     50㎕
 Transformation of BBa_J04450(3A) -> incubation

2010-09-19

From 2010 Biobrick
 plasmid with Cm antibiotic   -------------
  1-3C 1-3A pSB1C3
 plasmid with Amp antibiotic  ------------- 
  1-1K pSB1A3
 ↓
To transformate total five types, we seeded to plates.
 ↓
PCR products
 ↓
Gel extraction
 ↓  ← ADB buffer
ZYMO purification

1. To prepare 2ml tube(for ZYMO purification) + column, add the solution and operate centrifuge in 1 minute. Throw away left solution.

2. Same to mini-prep 

 * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1

3. NFW elution(10㎕) Add NFW and wait 1min. Operate centrifuge in 1min 4. Gel electrophoresis(each 1㎕) 

SOEing PCR
 ①T7/cI(OR2)-F-R
 ②T7/cI(OR1)-F-R
 ③Ptet-luxR-(OR2)
 ④Ptet-luxR-(OR1)
 ⑤Prom-luxR-(OR2)
 ⑥Prom-luxR-(OR1)
1. ①1㎕, ③3㎕
2. ②1㎕, ④4㎕
3. ①1㎕, ⑤1㎕
4. ②1㎕, ⑥1㎕

1. ①-③
2. ②-④
 --------------------------
 template    1㎕-4㎕
 primer Fwd  -
 primer Rev  -
 dNTP        5㎕
 10xbuffer   5㎕
 NFW         34㎕
 VentP       1㎕
 -------------------------
             50㎕

3. ①-⑤
4. ②-⑥
 -------------------------
 template      1㎕-1㎕
 primer Fwd    -
 primer Rev    -
 dNTP          5㎕
 10xbuffer     5㎕
 NFW           37㎕
 VentP         1㎕
 -------------------------
               50㎕

5. control
 -------------------------
 template      1㎕
 primer Fwd    5㎕
 primer Rev    5㎕
 dNTP          5㎕
 10xbuffer     5㎕
 NFW           28㎕
 VentP         1㎕
 -------------------------
               50㎕

PCR
 94°C – 5min
 94°C – 15sec     --
 52°C – 30sec       │- 10cycles
 72°C – 1min      --
 72°C – 10min
 ----------------
 4°C   stop

2010-09-20

Transformation 

BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
2-1 P    pSB1AK3
11:30 ~   DNA : 1㎕    XL10GkanR : 30㎕    SOC : 100㎕

Cm              Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3

liquid incubation(37°C) 7:15~
Mini-prep

ZYMO purification 

From PCR product from SOEing PCR (09-19)
1. Add PCR products(50㎕) and DNA binding buffer(200㎕) into 1.5mL tube(Commonly adding DNA binding buffer for double
amount of PCR products, but it must be 200㎕ at least). After that, mix this tube for a second with vortex and move
to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
2. centrifuge 1min → throwing away left solution
3. Same to mini-prep
 * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
4. NFW elution(10㎕)

↓
PCR 

PCR Condition
 94°C – 5min
 94°C – 15sec     --
 52°C – 30sec       │-10 cycles
 72°C – 1min      --
 72°C – 10min
 ----------------
 4°C   stop

After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
 -------------------------
 template      10㎕
 primer Fwd     5㎕
 primer Rev     5㎕
 dNTP           5㎕
 10xbuffer      5㎕
 NFW            19㎕
 VentP          1㎕
 -------------------------
                50㎕

Using primer
 1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 2. ②-④ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 3. ①-⑤ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 4. ②-⑥ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 5. control

Using template
 1. Ptet-LuxR-PT7/cI(OR2)-GFP
 2. Ptet-LuxR-PT7/cI(OR1)-GFP
 3. Prom-LuxR-Pt7/cI(OR2)-GFP
 4. Prom-LuxR-Pt7.cI(OR1)-GFP
 5. control

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

↓
Gel Electrophoresis 

Cm             Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3

↓
Liquid Incubation(37°C) 7:15~ 

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

Using primer
 Fwd : pSB1C3FA-F
 Rev : pSB1C3FA-R

Using template
 1. 1-3C(J04450,pSB3C5)
 2. pSB1C3
 3. 1-3A(J04450,pSB1C3)
 4. 1-1K(J04450,J63010)
 5. control(pCI-GFP)   primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev

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

↓
Gel Electrophoresis

Notebook

8/31 We've started experiment. First, we researched the property of T7 promoter.

9/5 We are researching promoter and inverters.

10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D
Retrieved from "http://2010.igem.org/Team:Chiba/Notebook_1"