Team:Osaka/Notebook

From 2010.igem.org

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(→Notebook)
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　　　　　　　LB3ml＋Amp3μl
　　　　　　　LB3ml＋Amp3μl
　　　　　　　16:20~　
　　　　　　　16:20~　
 +
 +
 +
===September 20===               
 +
#PCR
 +
::{| class="wikitable"
 +
|tube5  control
 +
:5xphusion  HF Buffer        4μl
 +
:10mM dNTP                0.4μl   
 +
:primer                      1μl
 +
:control template DNA      0.8μl   
 +
:phusion                  0.2μl
 +
:H2O                      13.6μl   
 +
:total                      20μl   
 +
 +
|thermal cycle
 +
:98℃    1min
 +
:98℃  5sec
 +
:60℃  15sec 
 +
:72℃  2min30sec        25cycle (boldface)
 +
:72℃  10min
 +
:4℃　　　∞
 +
 +
 +
|}
 +
 +
::{| class="wikitable"
 +
|tube1
 +
:GC Buffer                        4μl
 +
:dNTP                          0.4μl
 +
:10xprimer  Mix                2μl
 +
:template (10xA02)          1μl
 +
:phusion                          0.2μl
 +
:H2O                              12.4μl
 +
:total                                20μl
 +
 +
 +
|tube2
 +
:HF Buffer                        4μl
 +
:dNTP                          0.4μl
 +
:10xprimer  Mix                2μl
 +
:template (10xA02)          1μl
 +
:phusion                          0.2μl
 +
:MgCl2(1mM)                  4μl
 +
:H2O                                8.4μl
 +
:total                                20μl
 +
 +
|thermal cycle
 +
:98℃    1min
 +
:98℃  5sec
 +
:40℃  30sec 
 +
:72℃  50sec        30cycle (boldface)
 +
:72℃  10min
 +
:4℃　　　∞
 +
 +
 +
 +
 +
|}
 +
 +
::Control is good.
 +
::Sample is not good.
 +
:::problem of template and primer / thermal cycle and condition of reaction?
 +
 +
#PCR for checking primer
 +
:PCR
 +
::HF Buffer        32μｌ
 +
::10mM dNTP        3.2μｌ
 +
::Template(×10)    8μｌ
 +
::Phusion          1.6μｌ
 +
::DSW              112μｌ
 +
::total            156.8μｌ
 +
 +
:{| class="wikitable"
 +
|
 +
|(ⅰ)rev4fwd4
 +
|(ⅱ)rev3fwd3
 +
|(ⅲ)rev2fwd2
 +
|(ⅳ)pgsBrev1
 +
|-
 +
|Saka||1||3||5||7
 +
|-
 +
|Yasumoto||2||4||6||8
 +
|}
 +
 +
PCR（ⅰ）　 BlockA
 +
:{| class="wikitable"
 +
|5xPhusion HF Buffer 4μｌ   
 +
10mM dNTP  0.4μｌ
 +
<br>Template    1μｌ
 +
<br>primer    rev4    0.2μｌ
 +
<br>primer    fwd4      0.2μｌ
 +
<br>Phusion      0.2μｌ
 +
<br>DSW        14μｌ
 +
<br>total      20μｌ
 +
|thermal cycle
 +
:98℃  30sec
 +
:98℃  10sec
 +
:65℃  30sec 
 +
:72℃  30sec        35cycle(boldface)
 +
:72℃  10min
 +
:4℃　　　∞
 +
 +
 +
|}
 +
 +
PCR（ⅱ）　
 +
          5xPhusion HF Buffer        4μｌ
 +
            10mM dNTP                    0.4μｌ
 +
            Template (x10)                  1μｌ
 +
              primer    rev3                0.2μｌ
 +
              primer    fwd3                0.2μｌ
 +
              Phusion                        0.2μｌ
 +
              DSW                              14μｌ
 +
              total                                20μｌ
 +
thermal cycle
 +
98℃  30sec
 +
98℃  10sec
 +
70℃  30sec 
 +
72℃  30sec        35cycle (boldface)
 +
72℃  10min
 +
4℃　　　∞
 +
 +
PCR（ⅲ）　
 +
          5xPhusion HF Buffer        4μｌ
 +
            10mM dNTP                    0.4μｌ
 +
            Template (x10)                  1μｌ
 +
              primer    rev2                0.2μｌ
 +
              primer    fwd2                0.2μｌ
 +
              Phusion                        0.2μｌ
 +
              DSW                              14μｌ
 +
              total                                20μｌ
 +
thermal cycle
 +
98℃  30sec
 +
98℃  10sec
 +
70℃  30sec 
 +
72℃  30sec        35cycle (boldface)
 +
72℃  10min
 +
4℃　　　∞
 +
 +
PCR（ⅳ）　  BlockB
 +
          5xPhusion HF Buffer        4μｌ
 +
            10mM dNTP                    0.4μｌ
 +
            Template                          1μｌ
 +
              primer    pgsB                0.2μｌ
 +
              primer    rev1                0.2μｌ
 +
              Phusion                        0.2μｌ
 +
              DSW                              14μｌ
 +
              total                                20μｌ
 +
thermal cycle
 +
98℃  30sec
 +
98℃  10sec
 +
60℃  30sec 
 +
72℃  30sec        35cycle (boldface)
 +
72℃  10min
 +
4℃　　　∞
 +
 +
Check PCR fragments by each primers. 各primerによる断片作成チェック
 +
～primer erratum primerの正誤確認～
 +
98℃  30sec
 +
98℃  10sec
 +
X℃  30sec 
 +
72℃  30sec        35cycle (boldface)
 +
72℃  10min
 +
4℃　　　∞
 +
～Mix composition～
 +
          5xPhusion HF Buffer        4μｌ
 +
          10mM dNTPs                    0.4μｌ
 +
            Template(×10)                1μｌ
 +
              primer    1                      0.2μｌ
 +
              primer    2                      0.2μｌ
 +
              primer    3                      0.2μｌ
 +
              primer    4                      0.2μｌ
 +
              polymerase                    0.2μｌ
 +
              DSW                            13.6μｌ
 +
              total                                20μｌ
 +
　
 +
8. Electrophoresis
 +
Apply all sample 全量アプライ(dye 2μｌ、sample　20μｌ、Ladder　10μｌ)
 +
1%Agalose gel、100V　20min
 +
 +
  8  7  6  5  4  3  2  1  Ladder
 +
 +
  8 , 7→1      6 , 5→2        2 , 1→3
 +
Elution of each sample from gel and purification
 +
 +
3. pgsB  2nd PCR  +再々々々 1st PCR
 +
            HF Buffer                          4μｌ
 +
            dNTP                              0.4μｌ
 +
              primer    pgsB              0.2μｌ
 +
                            rev2                0.2μｌ
 +
              tube1                            0.2μｌ
 +
                tube2                            0.2μｌ
 +
              Phusion                        0.2μｌ
 +
              DSW                              14.6μｌ
 +
              total                                20μｌ
 +
 +
  protocol1
 +
  98℃  1min
 +
  94℃  15sec
 +
  50℃  30sec 
 +
  74℃  30sec        下３つを2cycle
 +
        　↓flanking  primer3
 +
  94℃  15sec
 +
  68℃  30sec
 +
  72℃  1min      上３つを25cycle
 +
  72℃  10min
 +
  4℃　　　∞
 +
 +
  protocol2  tube8  tube7
 +
  98℃  1min
 +
  98℃  10sec
 +
  50℃  30sec 
 +
  70℃  30sec        下３つを2cycle
 +
        　↓Hauking  primers
 +
  98℃  10sec
 +
  70℃  30sec
 +
  68℃  1min      上３つを25cycle
 +
  72℃  10min
 +
  4℃　　　∞
 +
　　　　　↓
 +
　　Electropjoresis
 +
 +
4. Electrophoresis
 +
dye 2μｌ、Sample　5μｌ、Ladder　4μｌ 
 +
1%Agalose gel、100V　20min
 +
 +
PCR1  PCR2  PCR3  Ladder
 +
 +
5. pgsB  1st PCR (part)
 +
      Mix composition
 +
          5xPhusion HF Buffer        4μｌ
 +
          10mM dNTPs                    0.4μｌ
 +
            Template                          1μｌ
 +
              primer    rev                  0.2μｌ
 +
              primer    fwd                  0.2μｌ
 +
              phusion                        0.2μｌ
 +
              DSW                            14μｌ
 +
              total                                20μｌ
 +
thermal cycle
 +
98℃  30sec
 +
98℃  10sec
 +
74℃  30sec 
 +
72℃  10sec        35cycle(boldface)
 +
72℃  10min
 +
4℃　　　∞
 +
 +
6. To liquid culture
 +
  004(C), 005(C)  ,006(K)  ,007(K)  008(K)  ,009(K)  ,019(A)
 +
To LB medium
 +
37 ℃ incubate          18:20~
 +
 +
New parts
 +
020(A)
 +
                pgsA
 +
 +
7. Ligation
 +
    pgsA  (Eco,Pst)d                    2μl
 +
    1-1C  (Eco,Pst)d                    2μl
 +
    10×T4 DNA ligase Buffer        2μｌ
 +
    T4 DNA ligase                        1μｌ
 +
    H2O                                      13μｌ
 +
    total                                        20μｌ
 +
        room  temp  10min  Ligation
 +
        80℃　 deactivtion by heat  20min
 +
 +
8. Transformation
 +
Competent cell②50μl＋LigationLigation product 2μl
 +
Inoculte A plate and incubt at 37oC
 +
2：00～
 +
 +
9. Electrophoresis
 +
All PCR product＋dye 2μｌ、Ladder　10μｌ
 +
1%Agalose gel、100V　25min
 +
 +
tube8  tube7  tube6  tube5  Ladder
 +
tube2  tube1  Ladder
 +
 +
tube1, 2  は300bpあたり
 +
tube5, 6, 7, 8 は740bpあたりのバンドを切り出し
 +
gel purification
 +
 +
10. Electrophoresis to check gel purification
 +
dye 2μｌ、sample 10μｌ、Ladder　10μｌ
 +
1%Agalose gel、100V　20min
 +
 +
tube8  tube7  tube6  tube5  tube2  tube1  Ladder

Revision as of 13:24, 6 October 2010

Calendar

July
S M T W T F S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
August
S M T W T F S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
September
S M T W T F S
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
October
S M T W T F S
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
November
S M T W T F S
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30











Notebook

July 29 (Thu)

Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura

  1. Safety lecture for junior members.
  2. Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).

July 31 (Sat)

Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka

  1. Meeting
    • Summer project schedule
    • List of genes to clone

August 2 (Mon)

Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka

  1. Culture medium preparation
    • LB agar plates (49 antibiotic-less plates)
    • LB liquid medium (500 ml)
  2. Competent cells preparation - Nojima Method
    • SOB medium (MgCl2 not yet added) -> stored at 4˚C
    • TB buffer -> stored at 4˚C
    • Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n

Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.

August 3 (Tue)

Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh

  1. Competent cells preparation (continued)
    • Preparation of glucose solution for making SOC medium.
    • Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
    • (Night) Transfer from pre-culture to growth culture.

August 4 (Wed)

Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh

  1. OD measurements throughout the day till required OD (0.3~0.7) was obtained.
  2. Completion of competent cells according to protocol.

August 5 (Thu)

Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino

  1. Transformation of Registry parts:
IDPart NameResistanceDescription
2-20J<bbpart>BBa_K118023</bbpart>AC. fermi endocellulase Cen A coding
2-20H<bbpart>BBa_K118022</bbpart>AC. fermi exocellulase Cex coding
1-2M<bbpart>BBa_B0034</bbpart>ARBS
1-13D<bbpart>BBa_B0010</bbpart>Aterminator
1-1D<bbpart>BBa_R0010</bbpart>Apromoter
1-18F<bbpart>BBa_E1010</bbpart>KRFP coding

Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.

August 6 (Fri)

Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh

  1. Colony check
    • All transformed cells produced colonies!
    • Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
  2. Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C

August 7 (Sat)

Attendance: Nakamura, Saka, Yasumoto, Takino

  1. Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
  2. Transformation of construction plasmids
IDPart NameResistanceDescription
1-1C<bbpart>pSB1A3</bbpart>Aconstruction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>)
1-3A<bbpart>pSB1C3</bbpart>C(" ")
1-5A<bbpart>pSB1K3</bbpart>K(" ")
  1. Meeting

August 8 (Sun)

Attendance: Nakamura, Yasumoto

  1. Colony check
    • All parts successfully transformed
  2. Transfer to LB culture medium

August 9 (Mon)

  1. Miniprep of 1-1C
  2. Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
    • (WHICH ENZYMES?)
  3. Gel electrophoresis of digests ("cut check")
    • 2-20H, 2-20J, 1-1C -> OK
    • 1-1D, 1-18F, 1-13D, 1-2M -> not cut (single band, MW approx. equal to vector + insert)
  4. Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
    • Yesterday's inoculated culture mediums contained the wrong antibiotics!
  5. Transformation of <bbpart>BBa_I13521</bbpart>, <bbpart>BBa_I13522</bbpart>, <bbpart>BBa_I13600</bbpart>, <bbpart>BBa_K204031</bbpart>, <bbpart>BBa_K204051</bbpart>, <bbpart>BBa_K204032</bbpart> from last year's stock

August 10 (Tue)

  1. Miniprep of 1-3A, 1-5A
  2. Restriction digests of 1-3A, 1-5A
  3. Gel electrophoresis
    1. 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
      • 1-3A, 1-5A -> OK; others -> not cut (again)
    2. 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
      • all parts not cut

August 11 (Wed)

  1. Miniprep of last year's parts transformed on Monday
  2. Gel electrophoresis of 1-1D, 1-18F, 1-13D, 1-2M (3rd repeat!!!)
    • all 4 not cut... AGAIN
    • so far all restriction digests involving XbaI seem to have failed; problem with enzyme stock?
    • will try with different set of restriction enzymes next week
  3. Sent miniprepped last year's parts to ECUST team in Shanghai, China

August 16 (Mon)

  1. Restriction digests
    • 1-13D,　1-2M,　1-1D,　1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
    • 2-20J with XbaI, PstI to check/confirm XbaI activity
  2. Gel electrophoresis of digests
    • (RESULTS?)
  3. Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D,　1-2M,　1-1D,　1-18F
    • 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added

August 17 (Tue)

  1. Mini-prep of 1-13D,　1-2M,　1-1D,　1-18F parts inoculated yesterday (2 of each)
  2. 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
    • 1-1D, 1-18F with EcoRI, SpeI
    • 1-13D, 1-2M with XbaI, PstI
    • (RESULTS?)
  3. Transformation of secretion tag parts using 25μl of competent cells each
IDPart NameResistanceDescription
2-22P<bbpart>BBa_K103006</bbpart>AOmpA outer membrane protein + linker
1-2J<bbpart>BBa_J32015</bbpart>A,KPelB leader sequence

August 18 (Wed)

iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda

August 19 (Thu)

  1. Transfer of 2-22P, 1-2J to solution culture
  2. Gel electrophoresis of digests from 'cut check' products from Tuesday
    • repeat run, but each digest together with undigested plasmid DNA)
    • 2% agarose gel instead of the usual 1%
    • (RESULTS?)
  3. Gel electrophoresis of 1-1D digest only
    • (RESULT?)
  4. Multiple restriction digests of 1-1D to check for problems at restriction sites
    • tried the following: EcoRI only; SpeI only; EcoRI + SpeI
  5. Night: miniprep of 2-22P, 1-2J inoculated in the morning

August 20 (Fri)

  1. Gel electrophoresis of 1-1D and its digests
    • (RESULTS?)
  2. 'Cut check' of parts miniprepped the night before
    • both 2-22P & 1-2J cut with XbaI, PstI
    • enzyme inactivation at 80℃, 20min
    • (RESULTS?)
  3. Restriction digest of 2-20J (WHICH ENZYMES?)
  4. Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
    • reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
    • reaction at room temperature for 10min; ligase inactivation at 80℃ for 20min
  5. Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix

August 21 (Sat)

  1. Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
    • we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
  2. 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
    • ligation product designated as <1>; Chloramphenicol resistance
    • same ligation mix composition as yesterday's
  3. Transformation of <1> with pre-incubation for 1.5hr instead of 1hr

August 22 (Sun)

  1. Transfer of <1> to culture solution; incubation at 30℃ (why??)
  2. Transformation of the following parts:
IDPart NameResistanceDescription
2-4A<bbpart>BBa_J63005</bbpart>Ayeast ADH1 promoter
F1N/AAbeta-galactosidase from Edinburgh team
F2N/ACRBS + F1
F3N/ACLac promoter + RBS + F1
    • O/N incubation at 37℃ as per normal

August 23 (Mon)

  1. Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
  2. Miniprep & 'Cut check' of <1>
    • cut at EcoRI, SpeI
    • gel run with DNA ladder, digested plasmid, undigested plasmid
    • (RESULTS?)
  3. Transfer of 3 more colonies of <1> to liquid solution (to store as glycerol stock - see Tue notes)
  4. Transformation of the following registry parts
IDPart NameResistanceDescription
2-2O<bbpart>J63003</bbpart>Ayeast Kozak sequence
3-11I<bbpart>K105027</bbpart>A'cyc100' minimal promoter

August 24 (Tue)

  1. Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
    • transfer to solution culture
  2. Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
    • (RESULTS)
  3. Transfer of F1 to solution culture (why?)
  4. Preparation of glycerol stock of cell culture containing <1> (why?)
    • 200ml of culture solution mixed with 100ml of 50% glycerol
    • stored at -80℃

August 25 (Wed)

  1. Miniprep of parts in solution culture: 2-2O, 3-11I, F1
  2. Cut check of 3-11I & F1 with EcoRI, SpeI
    • (RESULTS?)

August 26 (Thu)

  1. Transformation of <bbpart>BBa_K204022</bbpart>, <bbpart>BBa_K204025</bbpart>, and <bbpart>BBa_K204040</bbpart> to send to Shanghai ECUST team in China.

August 27 (Fri)

  1. Transfer of yesterday's transformed parts (all produced colonies) to solution culture
  2. Transformation of the following parts
IDPart NameResistanceDescription
1-1K<bbpart>BBa_J63010</bbpart>AProtein fusion vector (Silver standard)
1-1I<bbpart>BBa_J63009</bbpart>ALow copy protein fusion vector (Silver standard)
3-3G<bbpart>BBa_K157013</bbpart>A15aa glycine-serine linker (Freiburg standard)
    • using competent cells opened on 8/20
  2. Preparation of YPD yeast culture medium with the following recipe:
MiliQ water1 liter
Bacto tryptone20.0g2%
Bacto yeast extract10.0g1%
Glucose20.0g2%
    • pH was adjusted to 5.8
    • autoclaved before use
    • 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
  2. Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar

August 28 (Sat)

  1. Miniprep of parts in solution culture
  2. Restriction digest (for cut check) - 37℃ for 30min
    • 2-4A & 3-11I with EcoRI, SpeI
    • 2-2O with XbaI, PstI
    • K204022, K204025, K204040 wih EcoRI, PstI
  3. Gel electrophoresis of digests
    • Plasmids not detected for 2-4A & 3-11I - mistake during miniprep? culture duration too long, plasmid loss occurred?
  4. Transfer of the following parts to solution culture
    • 1-1K, 1-1I, 3-3G (yesterday's transformations)
    • 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)

August 29 (Sun)

  1. Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
  2. Restriction digests
    • for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
    • for assembly: 2-4A, 3-11I with EcoRI, SpeI (upstream parts)
  3. Gel electrophoresis for confirmation
    • Inserts seem to be present in all samples
  4. 3A assembly ligations:
    1. 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as <2>
    2. 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as <3>
    • 2-2O using XbaI, PstI digest from yesterday
    • 1-5A has Kan resistance
    • ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80℃
  5. Transformation of ligation products <2> and <3>

August 30 (Mon)

  1. Restriction digests for 3A assembly
    • 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
    • 2-20J (CenA), 2-20H (Cex), F1 (BglX) with XbaI, PstI
      • beta-glucosidase received from Edinburgh team temporarily designated BglX
  2. Gel electrophoresis of the digests to confirm inserts
    • all OK
  3. Transfer of <2> and <3> to solution culture (3 colonies each)

August 31 (Tue)

１．miniprep 

2-20-2-4-A     ←共に各3つずつ
2-20-3-11I

2-20-3-11I②ペレットが赤かった

☆Lysis Solutionの保存温度、取扱いミスによって今までの収量が悪かったのでは？ きちんと溶けていない状態で冷蔵していた→60℃に入れて溶かした （最初の段階で60℃に入れて溶かすとあるのでおそらく熱には大丈夫！）

New parts! 2-20-2-4-A　・・・ 002 2-20-3-11I ・・・003

002 

       yeast ADH1 promoter + kozak segnence(RBS for yeast)

003 

       cyc 100minimal promoter + kozak segnence (RBS for yeast)


２．制限酵素処理 

002①,003①
 プラスミド             2.5μｌ

　EcoRI 　　　　　1μｌ　 

 Spe I　　　　　　　　1μｌ　
10×NEbuffer2　　　5μｌ

100×BSA　　　　　　0.5μｌ 　H2O　　　　　　　 　 40μｌ 

 total                      50μｌ

37 ℃ incubate 30min

３．電気泳動 　dye 2μｌ、サンプル　10μｌ、Ladder　2μｌ 

1%Agalose gel、100V　20min

003①d 002①d Ladder

003①cutはできている

インサートが(18+103)bpよりも明らかに大きい

戻って見直すと2-20のインサートの長さがおかしい

ライゲーションはできている

2-20を再度培養しcut checkする

４．2-20の5つのコロニーをそれぞれLB培地に植える 　　　　　　　LB3ml＋Amp3μl 　　　　　　　16:20~　


September 20

  1. PCR
tube5 control
5xphusion HF Buffer 4μl
10mM dNTP 0.4μl
primer 1μl
control template DNA 0.8μl
phusion 0.2μl
H2O 13.6μl
total 20μl
thermal cycle
98℃ 1min
98℃ 5sec
60℃ 15sec
72℃ 2min30sec 25cycle (boldface)
72℃ 10min
4℃　　　∞


tube1
GC Buffer 4μl
dNTP 0.4μl
10xprimer Mix 2μl
template (10xA02) 1μl
phusion 0.2μl
H2O 12.4μl
total 20μl


tube2
HF Buffer 4μl
dNTP 0.4μl
10xprimer Mix 2μl
template (10xA02) 1μl
phusion 0.2μl
MgCl2(1mM) 4μl
H2O 8.4μl
total 20μl
thermal cycle
98℃ 1min
98℃ 5sec
40℃ 30sec
72℃ 50sec 30cycle (boldface)
72℃ 10min
4℃　　　∞



Control is good.
Sample is not good.
problem of template and primer / thermal cycle and condition of reaction?
  1. PCR for checking primer
PCR
HF Buffer 32μｌ
10mM dNTP 3.2μｌ
Template(×10) 8μｌ
Phusion 1.6μｌ
DSW 112μｌ
total 156.8μｌ
(ⅰ)rev4fwd4 (ⅱ)rev3fwd3 (ⅲ)rev2fwd2 (ⅳ)pgsBrev1
Saka1357
Yasumoto2468

PCR（ⅰ）　 BlockA

5xPhusion HF Buffer 4μｌ

10mM dNTP 0.4μｌ
Template 1μｌ
primer rev4 0.2μｌ
primer fwd4 0.2μｌ
Phusion 0.2μｌ
DSW 14μｌ
total 20μｌ

thermal cycle
98℃ 30sec
98℃ 10sec
65℃ 30sec
72℃ 30sec 35cycle(boldface)
72℃ 10min
4℃　　　∞


PCR（ⅱ）　 

          5xPhusion HF Buffer         4μｌ
           10mM dNTP                    0.4μｌ
           Template (x10)                  1μｌ
             primer    rev3                 0.2μｌ
             primer    fwd3                0.2μｌ
              Phusion                         0.2μｌ
              DSW                              14μｌ
             total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 70℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞

PCR（ⅲ）　 

          5xPhusion HF Buffer         4μｌ
           10mM dNTP                    0.4μｌ
           Template (x10)                  1μｌ
             primer    rev2                 0.2μｌ
             primer    fwd2                0.2μｌ
              Phusion                         0.2μｌ
              DSW                              14μｌ
             total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 70℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞

PCR（ⅳ）　 BlockB 

          5xPhusion HF Buffer         4μｌ
           10mM dNTP                    0.4μｌ
           Template                           1μｌ
             primer    pgsB                 0.2μｌ
             primer    rev1                0.2μｌ
              Phusion                         0.2μｌ
              DSW                              14μｌ
             total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 60℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞

Check PCR fragments by each primers. 各primerによる断片作成チェック ～primer erratum primerの正誤確認～ 98℃ 30sec 98℃ 10sec 

X℃   30sec

72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞ ～Mix composition～ 

          5xPhusion HF Buffer         4μｌ
         10mM dNTPs                     0.4μｌ
           Template(×10)                 1μｌ
             primer    1                      0.2μｌ
             primer    2                      0.2μｌ
             primer    3                      0.2μｌ
             primer    4                      0.2μｌ
             polymerase                    0.2μｌ
              DSW                            13.6μｌ
             total                                 20μｌ

　 8. Electrophoresis Apply all sample 全量アプライ(dye 2μｌ、sample　20μｌ、Ladder　10μｌ) 1%Agalose gel、100V　20min 

 8   7   6   5   4   3   2   1   Ladder

 8 , 7→1       6 , 5→2        2 , 1→3

Elution of each sample from gel and purification

3. pgsB 2nd PCR +再々々々 1st PCR 

           HF Buffer                          4μｌ
           dNTP                               0.4μｌ
             primer    pgsB              0.2μｌ
                           rev2                0.2μｌ
             tube1                             0.2μｌ
               tube2                             0.2μｌ
              Phusion                         0.2μｌ
              DSW                              14.6μｌ
             total                                 20μｌ

 protocol1
 98℃   1min
 94℃   15sec
 50℃   30sec   
 74℃   30sec        下３つを2cycle
        　↓flanking  primer3
 94℃   15sec
 68℃   30sec
 72℃   1min      上３つを25cycle
 72℃   10min
 4℃　　　∞

 protocol2   tube8  tube7
 98℃   1min
 98℃   10sec
 50℃   30sec   
 70℃   30sec        下３つを2cycle
        　↓Hauking  primers
 98℃   10sec
 70℃   30sec
 68℃   1min      上３つを25cycle
 72℃   10min
 4℃　　　∞

　　　　　↓ 　　Electropjoresis

4. Electrophoresis dye 2μｌ、Sample　5μｌ、Ladder　4μｌ 1%Agalose gel、100V　20min

PCR1 PCR2 PCR3 Ladder

5. pgsB 1st PCR (part) 

     Mix composition
          5xPhusion HF Buffer         4μｌ
         10mM dNTPs                     0.4μｌ
           Template                          1μｌ
             primer    rev                   0.2μｌ
             primer    fwd                   0.2μｌ
             phusion                         0.2μｌ
              DSW                            14μｌ
             total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 

74℃   30sec

72℃ 10sec 35cycle(boldface) 72℃ 10min 4℃　　　∞

6. To liquid culture 

 004(C), 005(C)  ,006(K)  ,007(K)  008(K)  ,009(K)  ,019(A)

To LB medium 37 ℃ incubate 18:20~

New parts 020(A) 

               pgsA

7. Ligation 

    pgsA  (Eco,Pst)d                    2μl
   1-1C  (Eco,Pst)d                     2μl
   10×T4 DNA ligase Buffer         2μｌ
   T4 DNA ligase                         1μｌ
    H2O                                       13μｌ
   total                                        20μｌ
        room  temp  10min  Ligation
        80℃　 deactivtion by heat   20min

8. Transformation Competent cell②50μl＋LigationLigation product 2μl Inoculte A plate and incubt at 37oC 2：00～

9. Electrophoresis All PCR product＋dye 2μｌ、Ladder　10μｌ 1%Agalose gel、100V　25min

tube8 tube7 tube6 tube5 Ladder tube2 tube1 Ladder

tube1, 2 は300bpあたり tube5, 6, 7, 8 は740bpあたりのバンドを切り出し gel purification

10. Electrophoresis to check gel purification dye 2μｌ、sample 10μｌ、Ladder　10μｌ 1%Agalose gel、100V　20min

tube8 tube7 tube6 tube5 tube2 tube1 Ladder


Retrieved from "http://2010.igem.org/Team:Osaka/Notebook"