Team:HSNU-TAIPEI/labnotes

Lab Notes

Extract the plasmid

  • Researcher: Zhao Ming-Cheng, Su Chin-Fong
  • Place: NTU
  • Date: September 14th, 2015

Procedure

We set them according to the following.

FIRST SECOND THIRD FORTH
Plasmid:Part1-QsrR(K1092000)-Terminator(B0015) 3μL Plasmid:Part1-QsrR(K1092000)-Terminator(B0015) 3μL Plasmid:Part2- RFP(E1010)-Terminator(B0015) Plasmid:Part2- RFP(E1010)-Terminator(B0015)
Buffer:M 1μL Buffer:M 1μL Buffer:M 1μL Buffer:M 1μL
Restriction enzyme: Xba1 0.25μL Restriction enzyme: Xba1 and Spe1 0.25μL each Restriction enzyme: Xba1 0.25μL Restriction enzyme: Xba1 and Spe1 0.25μL each
DdH2O:5.75μL DdH2O:5.50μL DdH2O:5.75μL DdH2O:5.50μL

And then do Gel electrophoresis.

Result

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Liquid culture

  • Researcher: Zhao Ming-Cheng, Su Chin-Fong
  • Place: NTU
  • Date: September 13th, 2015

Procedure

We take Part1-QsrR(K1092000)-Terminator(B0015) and Part2- RFP(E1010)-Terminator(B0015) which have been transformed to do liquid culture.

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Ligation

  • Researcher: Zhao Ming-Cheng, Su Chin-Fong
  • Place: NTU
  • Date: September 12th, 2015

Procedure

We have to do ligation one more time.

promoter(J23113)-RBS(B0034)-Laccase(K1092104)-RBS(B0034)-QsrR(K1092000)-Terminator(B0015)

promoter(J23113)-QsrR binding site-RBS(B0034)-RFP(E1010)-Terminator(B0015)

That is Part1-QsrR(K1092000)-Terminator(B0015) Part2- RFP(E1010)-Terminator(B0015)

After the Ligation,and then transform.

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Restriction Digest＆Ligation

  • Researcher: Chang Ko-Yu, Chu Yi-Chia
  • Place: Academia Sinica
  • Date: September 11th, 2015

Procedure

  1. Restriction Digest

    cyp1a2
    10X buffer 3
    DNA 5
    SpeI 0.6
    PstI 0.4
    ddH2O 21
    Total 30(μl)
    cyp1a2
    10X buffer 3
    DNA 5
    EcoRI-HF 0.5
    PstI 0.5
    ddH2O 21
    Total 30(μl)
    J23119
    10X buffer 3
    DNA 5
    XbaI 0.5
    PstI 0.5
    ddH2O 21
    Total 30(μl)
    B0015
    10X buffer 3
    DNA 5
    EcoRI-HF 0.5
    PstI 0.5
    ddH2O 21
    Total 30(μl)

  2. Run Gel(small well for check)

  3. Run Gel(big well for elute)

  4. Elute Fragment

  5. Ligation

    Psb1c3+cyp1a2
    T4 DNA Ligase 1
    Vector (psb1a2) 3
    nsert(cyp1a2 compsite) 14
    T4 DNA Ligase Buffer 2
    ddH2O 0
    Total 20μl
    23119+cyp1a2
    T4 DNA Ligase 1
    Vector (B0015) 3
    nsert(cyp1a2 ) 14
    T4 DNA Ligase Buffer 2
    ddH2O 0
    Total 20μl

    Place all sample in room temperature for 2hr

  6. Transform 10ul of the sample

    Nothing on the plate

Result

From left to right:1kb ladder,B0015+cyp1a2(Colony1-7),Psb1c3+cyp1a2(Colony1-4)

Cyp1a2-1＆Cyp1a2-5 are fault.

We deside to choose B0015+cyp1a2 Colony-2 Psb1c3+cyp1a2 compsite-3

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Extract the plasmid/Gel electrophoresis

  • Researcher: Zhao Ming-Cheng,Su Chin-Fong
  • Place: NTU
  • Date: September 11th, 2015

Procedure

Part1-QsrR(K1092000)-Terminator(B0015)

Part2- RFP(E1010)-Terminator(B0015)

After Extract the plasmid

We set them according to the following

FIRST SECOND THIRD FORTH
Plasmid:Part1-QsrR(K1092000)-Terminator(B0015) 3μL Plasmid:Part1-QsrR(K1092000)-Terminator(B0015) 3μL Plasmid:Part2- RFP(E1010)-Terminator(B0015) Plasmid:Part2- RFP(E1010)-Terminator(B0015)
Buffer:M 1μL Buffer:M 1μL Buffer:M 1μL Buffer:M 1μL
Restriction enzyme: Xba1 0.25μL Restriction enzyme: Xba1 and Spe1 0.25μL each Restriction enzyme: Xba1 0.25μL Restriction enzyme: Xba1 and Spe1 0.25μL each
DdH2O:5.75μL DdH2O:5.50μL DdH2O:5.75μL DdH2O:5.50μL

And then do Gel electrophoresis.

Result

From left to right

1~4 is the first group

5~8 is the second group

9 is 1kb marker

10~13 is the third group

14~17 is the forth group

But the length is wrong and there are Impurities in it.

So,the result is failed

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Liquid culture

  • Researcher: Zhao Ming-Cheng, Su Chin-Fong
  • Place: NTU
  • Date: September 10th, 2015

Procedure

We take Part1-QsrR(K1092000)-Terminator(B0015) and Part2- RFP(E1010)-Terminator(B0015) which have been transformed to do liquid culture.

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Check(Mini plasmid ＆ Run Gel)

  • Researcher: Chang Ko-Yu, Chu Yi-Chia
  • Place: Academia Sinica
  • Date: September 10th, 2015

Procedure

  1. Mini plasmid(14 colonies)

  2. Restriction Digest

    Psb1c3+cyp1a2 compsite(4sample)
    10X buffer 1
    DNA 8
    EcoRI-HF 0.5
    PstI 0.5
    ddH2O 0
    Total 10(μl)
    B0015+cyp1a2 (10sample)
    10X buffer 1
    DNA 8
    EcoRI-HF 0.5
    PstI 0.5
    ddH2O 0
    Total 10(μl)
  3. Run Gel

Result

From left to right:1kb ladder,B0015+cyp1a2(Colony1-7),Psb1c3+cyp1a2(Colony1-4)

Cyp1a2-1＆Cyp1a2-5 are fault.

We deside to choose B0015+cyp1a2 Colony-2 Psb1c3+cyp1a2 compsite-3

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Pick Colonies＆Culture bacteria

  • Researcher: Chang Ko-Yu, Chu Yi-Chia
  • Place: Academia Sinica
  • Date: September 9th, 2015

Procedure

  1. Pick different colonies to a new plate and a 5ml tube to culture them.
  2. Place all plate and tube in 37℃ overnight

Result

  1. plate
    2. Ps1c3+cyp1a2compsite B0015+cyp1a2
    Original plate
    Colonies we pick

  2. tube × 14

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Ligation

  • Researcher: Zhao Ming-Cheng, Su Chin-Fong
  • Place: NTU
  • Date: September 9th, 2015

Procedure

Then take to ligaiton，according to our circuit design is ：

promoter(J23113)-RBS(B0034)-Laccase(K1092104)-RBS(B0034)-QsrR(K1092000)-Terminator(B0015)

promoter(J23113)-QsrR binding site-RBS(B0034)-RFP(E1010)-Terminator(B0015)

That is,Part1-QsrR(K1092000)-Terminator(B0015) Part2- RFP(E1010)-Terminator(B0015)

After the Ligation,and then transform it

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Immobilization of E.coli

  • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen, Chen Szu-Hua, Chang Yu-Ting
  • Place: Fu Jen University Food Science Department
  • Date: September 8th, 2015

Material

  1. 3%SA(Alginate)
  2. 1.5%SA
  3. 1%CaCl2
  4. 0.3%Chitosan
  5. Beaker
  6. Syringe
  7. E.coli Dh5α
  8. LB broth
  9. eppendorf

Procedure

  • Step1: Inoculate E.coli into LB broth for 4hr.
  • Step2: Sterilize everything before putting it into the hood.
  • Step3: Mark each eppendorf.

  • Step4: Mix the solution as the amount written below.

    GROUP BACTERIAL LIQUID GEL
    SA 15ml 3%SA 60mL
    ACA 15ml 1.5%SA 60mL
    NO(CONTROL) 15ml LB Broth 60mL
  • Step5: Draw 0.5mL mixture by syringes and drop it into the forming solution (Immobilize E.coli by the method we try on September 1st, 2015).
  • Step6: Add the microcapsules into each eppendorf.
  • Step7: Keep the microcapsules in different temperatures (25℃/4℃/-18℃)

    • Result

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    Transformation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: September 8th, 2015

    Procedure

    Transform all sample(20μl) we ligase yesterday (Ps1c3+cyp1a2compsite) (B0015+cyp1a2)

    Result

    Ps1c3+cyp1a2compsite B0015+cyp1a2
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    Ligation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: September 7th, 2015

    Procedure

    1. Total:20μl

    2. Vector:Insert=1:3

      Psb1c3+cyp1a2 compsite
      T4 DNA Ligase 1
      Vector (psb1a2) 7
      Insert(cyp1a2 compsite) 10
      T4 DNA Ligase Buffer 2
      ddH2O 0
      Total 20(μl)
      B0015+cyp1a2
      T4 DNA Ligase 1
      Vector (B0015) 7
      Insert(cyp1a2) 10
      T4 DNA Ligase Buffer 2
      ddH2O 0
      Total 20(μl)
    3. Place the sample at 16℃ overnight
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    Elute DNA

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: September 7th, 2015

    Procedure

    1. Add 500μl DF Buffer into the tube with gel.
    2. 55-60℃ for 10-15 min (the gel completely dissolve)
    3. Cool in room temperature
    4. Put the DF Colume in the 2ml Collection
    5. Add 800μl sample in the DF Colume
    6. Centrifuge at 13000xg for 30sec
    7. Discard the flow-throgh
    8. Add 400μl W1 Buffer in the DF Colume
    9. Centrifuge at 13000xg for 30sec
    10. Discard the flow-throgh
    11. Add 600μl Wash Buffer in the DF Colume
    12. Centrifuge at 13000xg for 3min
    13. Put DF Colume to the 1.5ml tube
    14. Add 30μl Elution
    15. Place it for 2 min
    16. Centrifuge at 13000xg for 2min
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    Growth Curve

    • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen, Chen Szu-Hua, Chang Yu-Ting
    • Place: Fu Jen University Food Science Department
    • Date: September 6th, 2015

    Purpose

    To find out the time and temperature of E.coli’s culture to grow until our goal of bacterial count.

    Material

    1. E.coli
    2. LB agar
    3. 0.85% saline
    4. Tube
    5. Dish

    Procedure

    Step1: Draw one-hundredth amount of bacteria liquid into liquid medium (Inoculation) and put it into 37℃ incubator.

    Step2: Aliquot saline to each tube (9mL/tube).

    Step3: Mark every dish.

    Step4: Take out the bacteria liquid at 4 / 4.5 / 5 / 5.5 / 6 / 6.5 / 7 / 7.5 /8 hours and start serial dilution.

    Step5: Draw 1mL bacteria liquid into the first tube and pipet it.

    Step6: Exchange a tip, draw 1mL from the first tube and add it into the next tube.

    Step7: Repeat Step6 until the seventh tube.

    Step8: Exchange a tip and draw 1mL to a matched dish from the seventh to the first tube.

    Step9: Pour LB agar to each dish (10mL/dish) and shake it by an infinity sign.

    Step10: After LB agar’s solidification, place these dishes upside down in the 37℃ incubator.

    Result

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    Activation of Culture

    • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen
    • Place: Fu Jen University Food Science Department
    • Date: September 4th, 2015

    Material

    1. E.coli(DH5α、CmR J22102)
    2. Tube
    3. LB broth
    4. Inoculating loop
    5. Alcohol Burner

    Procedure

    Step1:Draw 10mL LB broth with 5mL pipet into a tube (total4).

    Step2: Scrape a colony with an inoculating loop sterilized by alcohol burner.

    Step3: Add the colony into the tube (two for each strain).

    Step4:Keep it in 37℃ incubator.

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    Restriction Digest

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: September 4th, 2015

    Procedure

  • Total:30μl

  • Enzyme:≤5%(1.5μl)

    Buffer U(DNA/mg in 50μl Temper
    EcoRI-HF 2.1[100] 20 37℃
    SpeI 2.1[100] 10 37℃
    PstI

    3.1[100]

    2.1[75]

    		 20 37℃
    XbaI 2.1[100] 20 37℃

    We descide to use 2.1 Buffer.

  • B0015(75.5ng/μl)
    10x buffer 3
    DNA 13.5
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 12.8
    Total 30μl
    B0034(208ng/μl)
    10x buffer 3
    DNA 4.8
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 21.1
    Total 30μl
    B0032(350.5ng/μl)
    10x buffer 3
    DNA 2.9
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 23.1
    Total 30μl
    E0040(188.5ng/μl)
    10x buffer 3
    DNA 5.3
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 20.7
    Total 30μl
    CYP1A2(100ng/μl)
    10x buffer 3
    DNA 10
    EcoRⅠ-HF 0.4
    XbaI 0.6
    ddH2O 16
    Total 30μl
    CYP1A2 COMPOSIE(10ng/μl)
    10x buffer 3
    DNA 10
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 16
    Total 30μl
    J23119(220ng/μl)
    10x buffer 3
    DNA 4.5
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 21.5
    Total 30μl
    E1010(162ng/μl)
    10x buffer 3
    DNA 6.2
    EcoRⅠ-HF 0.4
    XbaI 0.6
    ddH2O 19.8
    Total 30μl
    B0032(350.5ng/μl)
    10x buffer 3
    DNA 2.9
    EcoRⅠ-HF 0.5
    XbaI 0.5
    ddH2O 23.1
    Total 30μl

  • Result

    • We only succeed B0015 ,B032, CYP1A2, and CYP1A2 COMPOSIE

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    Survival

    • Researcher: Zhao Ming-Cheng, Su Chin-Fong
    • Place: NTU
    • Date: September 4th, 2015

    Procedure

    First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr.

    We divided two categories A and B.

    A:

    Take 80μL into 2ml LB broth x 6 tubes and then culture 1 hr.

    After 1hr,add 20μL benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))

    And add 20μL DMSO into the other tubes.Then,culture for 3hr.

    After 3hr,dilute the broth to 10-6

    And take 200μL to spread the plate.

    B:

    Take 80μL into 2ml LB broth in a tube And then culture 1 hr.

    After 1hr, put them into 6 tubes equally.

    Dilute the broth to 5×10-4

    Add 0.4μL benzo[a]pryene(2×10-4) in three tubes.

    Add 0.4μL DMSO in the other three tubes.

    Go to 37 degree Celsius shaking for 10min.

    Take 200μL to spread the plate.

    Result:

    According to the result, Beno[a]pryene does not affect E.coli’s survival.

    But Category B is failed because its number of colony is too much.

    Benzo[a]pryene Category A

    Control Category A

    Benzo[a]pryene CategoryB

    Control CategoryB

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    Preprocess of Growth Curve

    • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen
    • Place: Fu Jen University Food Science Department
    • Date: September 3rd & 4th, 2015

    Material

    1. LB agar
    2. 0.85% saline
    3. Tube
    4. Dish

    Procedure

    1. Solid medium

      Step1:Get certain weight of LB broth (25g/L) and agar (1.5%)

      Step2: Add the powder into the serum bottle with a wash bottle.

      Step3: Add certain amount of ddH2O into the serum bottle.

      Step4:Dissolve the solution with a spinbar.

      Step5:Sterilization for 1.5hr.

      Step6:Distribute to each dish (10mL/dish).

    2. Buffer solution

      Step1: Get certain weight of saline (0.85%)

      Step2: Add the powder into the serum bottle with a wash bottle.

      Step3: Add certain amount of ddH2O into the serum bottle.

      Step4:Sterilization for 1.5hr.

      Step5:Distribute to each tube (9mL/tube).

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    Pre-test of Gel Entrapment

    • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen
    • Place: Fu Jen University Food Science Department
    • Date: August 31th & September 1st 2015

    Purpose

    1. Make sure if the procedure from others’ reference fits our experiment.
    2. Compare the different effect of using syringe and pipet.

    Material

    1. PVA(Polyvinyl alcohol)
    2. SA(Alginate)
    3. BA(Boric acid)
    4. CaCl2
    5. Chitosan
    6. Sodium citrate(Na3C6H5O7•2H2O)
    7. Beaker
    8. Syringe
    9. Pipet

    Procedure

    • PVA-SA

      Step1: Pour some solution into beakers.

      Step2: Draw certain amount of 8%PVA-1%SA and add it into 3%BA-1%CaCl2.

    • SA

      Step1: Pour some solution into beakers.

      Step2: Draw certain amount of 3%SA and add it into 1%CaCl2.

    • ACA

      Step1: Pour some solution into beakers.

      Step2: Draw certain amount of 1.5%SA and add it into 100mmol/LCaCl2

      Step3: Throw the microcapsule into 0.3%Chitosan.

      Step4: Throw the microcapsule into 0.15%SA.

      Step5: Use syringe to inject sodium citrate in the microcapsule.

    Result

    1. Different method of entrapment

      PVA-SA SA ACA
      White, Teardrop-shaped colorless (bluish), sphere Bluish, sphere

    2. Step5 of ACA is infeasible, so we decide to delete this step.

    3. Comparison of syringe and pipet:

      syringe pipet
      Consistent size, faster, without bubble Vary in size, slow, with bubble
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    Four-phase Streaking method

    • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen
    • Place: Fu Jen University Food Science Department
    • Date: August 31th, 2015

    Purpose

    Plate streaking is an important and essential technique in molecular biology. Streaking allows the selection of a single colony from an original mixture of colonies. It also allows for the selection of one single colony from an original plate to be grown up as many colonies on a new plate.

    Material

    1. E.coli(DH5α)
    2. Solid medium(LB agar)
    3. Inoculating loop
    4. Alcohol Burner

    Procedure

    Step1: Sterilize the inoculating loop on an alcohol burner flame and allow it to cool for a few seconds.

    Step2: Using the loop, streak the first section of the plate using tight sweeping lines that stay within that section.

    Step3: Sterilize the loop and allow it to cool in the air for 15 seconds. Touch the loop to an unused edge of the agar surface to cool it completely before continuing.

    Step4: Pull the loop through the previous streak one or two times to re-inoculate the loop with cells.

    Step5: Streak section 2 of the plate, avoiding section 1 after the first 1-2 streaks and trying not to overlap the streaks.

    Step6: Repeat Step3,4,5 for two more times.

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    Gel electrophoresis Gel extraction

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 30th, 2015

    Procedure

    Today,we get the plasmid which be sent from the United States.

    They are:

    part1:promoter(J23113)-RBS(B0034)-Laccase(K1092104)-RBS(B0034)-

    Part2:promoter(J23113)-QsrR binding site-RBS(B0034)-

    We set them according to the following

    FIRST SECOND THIRD FORTH
    Plasmid:part1 4μL Plasmid:part1 4μL Plasmid:part2 4μL Plasmid:part2 4μL
    Buffer:M 4μL Buffer:M 4μL Buffer:10xM 4μL Buffer:M 4μL
    Restriction enzyme: Xba1 0.4μL Restriction enzyme: Xba1 and Pst1 0.4μL each Restriction enzyme: Xba1 0.4μL Restriction enzyme: Xba1 and Pst1 0.4μL each
    DdH2O:31.2μL DdH2O:30.8μL DdH2O:31.2μL DdH2O:30.8μL

    Put in 37 degree Celsius for 2 hr and then do Gel electrophoresis.

    Result

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    Gel extraction

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 29th, 2015

    Procedure

    We take QsrR(K1092000)-Terminator(B0015) and RFP(E1010)-Ter(B0015) to do gel extraction.

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    Plasmid Extraction

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: August 27th, 2015

    We extract the plasmid of B0015

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    Activation of Culture

    • Researcher: Leng Yi-Yan, Chang Chun-chieh, Lee Ming-Jhen, Chen Szu-Hua, Chang Yu-Ting
    • Place: Fu Jen University Food Science Department
    • Date: August 24th & 25th, 2015

    Material

    1. PVA(Polyvinyl alcohol)
    2. SA(Alginate)
    3. BA(Boric acid)
    4. CaCl2
    5. Chitosan
    6. Sodium citrate(Na3C6H5O7•2H2O)
    7. Electronic balance
    8. Spatulas
    9. Beaker
    10. ddH2O
    11. Volumetric flask
    12. Mixing rod
    13. Hotplate
    14. Serum bottle

    Procedure

    Step1: Get certain weight of chemicals with spatulas by an electronic balance.

    Step2: Use a volumetric flask to prepare a certain concentration of solution.

    Step3: Dissolve the solution with a stirring rod. (if not dissolving, use the hotplate until the solution becomes clear)

    Result

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    Send the DNA sequencing

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: August 20th, 2015

    Procedure

    1. Add 223.3 μl and 291.1 μl water to two different primer, and the concentration of the two primer is 100uM. Then put the two primer at 55 degree Celsius for fifteen minutes
    2. Diluted the two primer 10-fold to a concentration of 10μM for use
    3. 1μg of the plasmid (220ng/μl) 4.5(μl)
      ddH2O 5.5(μl)
      Total 10(μ)l
      Primer 2(μl)
      12(μ)l
    4. Send the DNA sequencing
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    DH5α-Pretest

    • Researcher: Zhao Ming-Cheng, Su Chin-Fong
    • Place: NTU
    • Date: August 20th, 2015

    Procedure

    Because we must test E.coli’s Survival in the environment there is benzo[a]pryene by counting the colonies.

    First we test how much concentration is the best.

    1. culture (8/17)

      STEP1:take 1μL DH5α to spread the plate(no Antibiotic)

      STEP2:put in 37 degree Celsius 12~16hr

    2. liquid culture(8/18)

    3. (8/19)

      STEP1:put 80μL into 2ml LB broth

      STEP2:recovering

      STEP3:After 2hr,dilute it to 10-4,10-5,10-6,10-7,and then go to spread the plate (no Antibiotic)

      STEP4:After 4hr dilute it to 10-4,10-5,10-6,10-7,and then go to spread the plate (no Antibiotic),6hr and 8hr Similarly

      STEP5:Take 200μL out from the tube and spread the plate(AMP+)

      STEP6: put in 37 degree Celsius 12~16hr

    RESULT:

    The number of the colonies in the AMP+ plate is zero.

    According to the result, 2hr 10-5 and 4hr 10-6 is the best.

    2hr plate from left to right is 10-4,10-5,10-6,10-7

    4hr plate from left to right is 10-4,10-5,10-6,10-7

    AMP+ Plate

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    Extract the plasmid

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 19th, 2015

    Procedure

    Extract QsrR(K1092000)-Ter(B0015)

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    Gel electrophoresis

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 19th, 2015

    Procedure

    We take QsrR(K1092000)- Terminator(B0015) to gel electrophoresis

    We set them according to the following

    FIRST SECOND
    Plasmid:QsrR-Ter 3μL Plasmid:QsrR-Ter 3μL
    Buffer:cutsmart 1μL Buffer:cutsmart 1μL
    Restriction enzyme:Xbal1 0.25μL Restriction enzyme:Xbal1 Pst1 0.25μL
    DdH2O:5.75μL DdH2O:5.50μL

    RESULT:

    Success!!

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    DH5α-Pretest

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 18th, 2015

    Because we must test E.coli’s Survival in the environment there is benzo[a]pryene by counting the colonies,First we test how much concentration is the best.

    1. culture (8/17)

      STEP1:take 1μL DH5α to spread the plate(no Antibiotic)

      STEP2:put in 37 degree Celsius 12~16hr

    2. liquid culture(8/18)

    3. (8/19)

      STEP1:put 80μL into 2ml LB broth

      STEP2:recovering

      STEP3:After 2hr take 200μL out to spread the plate (no Antibiotic)

      STEP4:After 4hr take 200μL out to spread the plate (no Antibiotic), 6hr and 8hr Similarly

      STEP5:put in 37 degree Celsius 12~16hr

    RESULT:

    We hope the number of colonies between 30~300,but the result is the colonies is too much!!

    Show More

    Liquid culture

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 18th, 2015

    Procedure

    Culture QsrR(K1092000)-Ter(B0015)

    Show More

    Extract the plasmid

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 16th, 2015

    Procedure

    Extract RFP(E1010)-Ter(B0015)

    Show More

    Liquid culture

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 15th, 2015

    Procedure

    Culture RFP(E1010)-Ter(B0015)

    Show More

    8/14/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 14th, 2015

    We successfully draw out the plasmid from E.coli.

    Show More

    Extract the plasmid

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 14th, 2015

    Procedure

    Extract QsrR(K1092000) plasmid

    Show More

    Ligation&Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 14th, 2015

    Procedure

    Ligation and Transform RFP(E1010)-Terminator(B0015)

    Show More

    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 14th, 2015

    Procedure

    Transform QsrR(K1092000)

    Show More

    8/13/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 13th, 2015

    We pick up the single column and incubate it.

    Show More

    Liquid culture

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 12th, 2015

    Procedure

    Culture RFP(E1010) and Terminator(B0015)

    Show More

    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 12th, 2015

    Procedure

    Transform QsrR(K1092000)

    Show More

    Gel electrophoresis

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 12th, 2015

    Procedure

    We take QsrR(K1092000) to gel electrophoresis

    We set them according to the following

    FIRST SECOND
    Plasmid:3μL Plasmid:3μL
    Restriction enzyme:EcoR1 0.25μL Restriction enzyme:EcoR1 0.25μL Spel1 0.25μL
    Buffer(smartcut):1μL Buffer(smartcut):1μL
    ddH2O:5.75μL ddH2O:5.50μL

    RESULT:

    The picture is too blurry.

    We think that it is because the concentration is too low.

    So we must transform one more time.

    Show More

    Transformation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: August 12th, 2015

    We transform J22106 this time.

    Show More

    8/12/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 12th, 2015

    We get another RBS B0032 and transform it.

    Show More

    Send the second DNA sequencing

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: August 11th, 2015

    Procedure

    1. Add 223.3 μl and 291.1 μl water to two different primer, and the concentration of the two primer is 100uM. Then put the two primer at 55 degree Celsius for fifteen minutes
    2. Diluted the two primer 10-fold to a concentration of 10μM for use
    3. 1μg of the plasmid (220ng/μl) 4.5(μl)
      ddH2O 5.5(μl)
      Total 10(μ)l
      Primer 2(μl)
      12(μ)l
    4. Send the DNA sequencing
    Show More

    8/11/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 11th, 2015

    The transforming is failed.

    Show More

    Gel electrophoresis

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 11th, 2015

    Procedure

    STEP1:test RFP’s andTerminator’s concentration

    STEP2:regulate their concentration to 1000np/μL

    STEP3:We set them according to the following

    FIRST SECOND THIRD FORTH
    Plasmid:RFP 4μL Buffer:cutsmart 4μL Restriction enzyme: EcoR1 0.4μL DdH2O:31.2μL
    Plasmid:RFP 4μL Buffer:cutsmart 4μL Restriction enzyme: EcoR1 and Spe1 0.4μL each DdH2O:30.8μL
    Plasmid:Ter 4μL Buffer:cutsmart 4μL Restriction enzyme: EcoR1 0.4μL DdH2O:31.2μL
    Plasmid:Ter 4μL Buffer:cutsmart 4μL Restriction enzyme: EcoR1 and Spe1 0.4μL each DdH2O:30.8μL

    RESULT:

    Nothing

    The result is failed

    Show More

    Extract the plasmid

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 11th, 2015

    Procedure

    Extract QsrR(K1092000) plasmid

    Show More

    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 11th, 2015

    Procedure

    Transform RFP(E1010) and Terminator(B0015)

    Show More

    Liquid culture

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 10th, 2015

    Procedure

    Culture QsrR(K1092000)

    Show More

    8/10/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 10th, 2015

    We get the part B0034 and transform it.

    Show More

    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 9th, 2015

    Procedure

    Transform QsrR(K1092000)

    Show More

    8/6/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 6th, 2015

    Use the enzyme to check the part length correct or not one more time and determine that we success.

    Show More

    8/5/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 5th, 2015

    Use the enzyme to check the part length correct or not.

    Show More

    Extract the plasmid

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 5th, 2015

    Extract RFP(E1010) and Terminator(B0015) plasmid

    Show More

    Liquid culture

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 4th, 2015

    Procedure

    culture RFP & Terminator(B0015)

    Show More

    Confirm the first DNA sequencing and Order the primer of second DNA sequencing

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: August 4th, 2015

    the primer of second DNA sequencing

    TACAGAGTTCTTGAAGTGGTGGCC

    TTTAAAGAAAAAGGGCAGGGTGGT

    Show More

    8/4/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: August 4th, 2015

    We successfully draw out the plasmid.

    Show More

    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: August 3rd, 2015

    Procedure

    Transform RFP & Terminator(B0015)

    Show More

    Plasmid Extraction

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: July 30th, 2015

    We extract the plasmid of B0034 and J23119 and test their concentration

    B0034 J23119
    concentration 208 ng/μl 220 ng/μl
    Show More

    7/29/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 29th, 2015

    We fell to draw out the plasmid.

    Show More

    Transformation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: July 28th, 2015

    We transform J23119 this time.

    Show More

    7/28/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 28th, 2015

    We incubate our E.coli.

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    7/27/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 27th, 2015

    We purify the gel , ligate the part, and transform into the E.coli.

    Show More

    7/24/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 24th, 2015

    We use gel electrophoresis to cut our part. Than we purify our gel.

    Show More

    7/23/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 23rd, 2015

    We draw out the plasmid from single column by plasmid mini kit.

    And start to use restriction enzyme to cut the part E1010 and B0015.

    Show More

    7/22/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 22nd, 2015

    We draw out the plasmid from single column by plasmid mini kit but failed.

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    7/21/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 21st, 2015

    We get the part B0015.

    Draw out the plasmid from single column by plasmid mini kit.

    We are going to do the combine the part in time but our plasmid disappeared.

    Show More

    7/17/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 17th, 2015

    Using gel electrophoresis again and check the part have problem.

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    7/16/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 16th, 2015

    We use gel electrophoresis to cut our part, and check the part length and determine that the part B0030 which we have has a mistake.

    Show More

    7/15/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 15th, 2015

    Draw out the plasmid from single column by plasmid mini kit{E0040,B0030,E1010,K896008}

    Show More

    7/14/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 14th, 2015

    We pick single column, and incubate in another tube.

    Show More

    7/13/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: July 13th, 2015

    We get “k89608” part and transform it.

    Show More

    Transformation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: July,2nd, 2015

    Because transformation we did on 6/11 was fail, this week, we did transformation again.

    In order to find the cause, we did two control group. One is J23102 the other is the kit provided. And we transform B0034 this time.

    Show More

    Plasmid Purification

    • Researcher: Shen Yu-Chun, Chen Sheng-Yuan
    • Place: NTUCM
    • Date: June 18th, 2015

    Material:

    1. 70 % Ethanol
    2. Isopropanol
    3. 50 ml centrifuge tubes
    4. Plasmid DNA Midi Kit
    5. pSB1C3-BBa_B0030 in DH5α
    6. pSB1C3-BBa_E1010 in DH5α
    7. pSB1A2-BBa_E0040 in DH5α
    8. pSB1A2-BBa_B0010 in DH5α

    Notes:

    1. Add RNase A to PL1 Buffer and store at 4℃
    2. Warm PL2 Buffer in a 37℃ waterbath
    3. Use 100~150 ml of bacterial culture for Midi Kit

    Protocol:

    1. Flick a PM Column 2~3 times, then place the PM Column on a conical flask.
    2. Equilibrate the PM Column by applying 5 ml of PL4 Buffer by gravity flow
    3. Harvest the bacterial culture 100 ml by centrifuge at 6000 x g for 10 mins
    4. Add 5 ml of PL1 Buffer to resuspend the cell pellet by vortexing or pipetting
    5. Add 5 ml of PL2 Buffer and mix gently by inverting the tube 10 times. DO NOT VORTEX!!!
    6. Place for 10 mins at room temperature until lysate clears
    7. Add 5 ml of PL3 Buffer and mix immediately by inverting the tube 10 times. DO NOT VORTEX!!!
    8. Centrifuge at 15000g for 20 minutes or filtered with a filter paper
    9. Apply the supernatant from step 8. to the equilibrated PM Column and allow it to flow by gravity flow.
    10. Wash the PM Column by using PL5 Buffer 15 ml and allow the column empty by gravity flow.
    11. Discard the filtrate.
    12. Place PM Column in a clean centrifuge and apply 10 ml of PL6 Buffer to elute DNA by gravity flow
    13. Using eluates from step 12. Add 7.5 ml of room temperature isopropanol. Vortex well and let the mixture sit for 2 minutes.
    14. Centrifuge at 12500 x g for 30 minutes.
    15. Remove the supernatant, then add 3 ml of 70% ethanol to wash.
    16. Centrifuge at 12500 x g for 5 minutes, then dry ethanol at room temperature.
    17. Apply 0.2 ml of TE Buffer to resuspend DNA.
    Show More

    6/18/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: June 18th, 2015

    We get “B0030, E0040, E1010”part,and transform them.

    Show More

    Ag+ adsorb on Au-NPs affect with different heavy metal ( Cu, Hg )

    • Researcher: Lin Sheng, Chu Wei-Min
    • Place: NTU Chemistry
    • Date: June 18th, 2015

    Procedure

    1. total:400 μL
    2. material:
      • 13nm Au-NPs 1.5mM,Pb 1mM100μM10μM
      • Cd 1mM100μM10μM,H7 Tris-Borate buffer 100mM,
      • AR 100μM,H2O21mM

    3. Caclulate:

      Pb2+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 220μL 20μL 40μL 0μL 40μL 40μL
      100μM 40μL 180μL 20μL 40μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 180μL 20μL 40μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 180μL 20μL 40μL 40μL(10μM→1μM) 40μL 40μL
      Cd2+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 220μL 20μL 40μL 0μL 40μL 40μL
      100μM 40μL 180μL 20μL 40μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 180μL 20μL 40μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 180μL 20μL 40μL 40μL(10μM→1μM) 40μL 40μL
      Pb2+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 260μL 20μL 0μL 0μL 40μL 40μL
      100μM 40μL 220μL 20μL 0μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 220μL 20μL 0μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 220μL 20μL 0μL 40μL(10μM→1μM) 40μL 40μL
      Cd2+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 260μL 20μL 0μL 0μL 40μL 40μL
      100μM 40μL 220μL 20μL 0μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 220μL 20μL 0μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 220μL 20μL 0μL 40μL(10μM→1μM) 40μL 40μL

    4. Result:

      We put all the result into fluorescent reader.

      This one is stand for Pb+.

      This one is stand for Cd2+.

    Show More

    6/15/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: June 15th, 2015

    We do the ligation, and then transform the plasmid into E.coli

    Mixing with LB broth, we smear the solid on the plate.

    Show More

    6/14/2015 Cadmium

    • Researcher: Chang I, Chi Ying-Cheng
    • Place: NTNU
    • Date: June 14th, 2015

    This is our first time do the experience.

    We are going to use the restriction enzyme to cut the sample part {E GFP} and add a part by ligation.

    So first, we use enzyme to cut the plasmid, and use gel electrophoresis to cut our part.

    Than we purify our gel.

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    Transformation

    • Researcher: Shen Yu-Chun, Chen Sheng-Yuan
    • Place: NTUCM
    • Date: June 11th, 2015

    Material:

    1. E.coli DH5α (ps: E.coli should be kept on the ice at all time)
    2. Plasmid : pSB1A2-BBa_E0040(Ampicillin-resistant) GFP
    3. Plasmid : pSB1C3-BBa_B0010(Chloramphenicol-resistant) Terminator
    4. Plasmid : pSB1C3-BBa_B0030(Chloramphenicol-resistant) RBS
    5. Plasmid : pSB1C3-BBa_E1010(Chloramphenicol-resistant) RFP
    6. LB broth (10 g /L Tryptone, 5 g/L Yeast extract, 5 g/L NaCl )
    7. LB plate(LB broth + 1% agar)
    8. Antibiotics : Ampicillin & Chloramphenicol (100μg/mL for each)

    Protocol:

    1. Add 0.5μL of plasmid into 100μL of E.coli and mix by tapping the tube.(ps: the quantity of plasmid is dependent on bacterial competency)
    2. Put the tube on the ice for 20~30mins.
    3. Remove from the ice and put the tube to the 42℃ hot bath for 45 sec.
    4. After heat shock, put the tube into the ice IMMEDIATELY for 2 mins.
    5. Add 500 μL of LB broth into the tube and incubate for 30~60 mins at 37℃
    6. Take out the tube and put into 6000 rpm centrifuge for 5 mins.
    7. Remove supertant and remain about 100μL of LB to resuspend
    8. Spread onto LB plate containing the appropriate antibiotic
    9. Incubate overnight for 12~16 hrs at 37℃.
    Show More

    Transformation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: June 11th, 2015

    This week, we did transformation.

    We transform B0034, B0015, I765001, I732005.

    Show More

    Ag+ adsorb on Au-NPs affect with different heavy metal ( Cu, Hg )

    • Researcher: Lin Sheng, Chu Wei-Min
    • Place: NTU Chemistry
    • Date: June 4th, 2015

    Procedure

    1. total:400 μL
    2. material:
      • 13nm Au-NPs 15mM,Cu 1mM100μM10μM
      • Hg 1mM100μM10μM,pH7 Tris-Borate buffer 100mM
      • AR 100μM,H2O2 1mM
    3. Caclulate:
      Cu2+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 220μL 20μL 40μL 0μL 40μL 40μL
      100μM 40μL 180μL 20μL 40μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 180μL 20μL 40μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 180μL 20μL 40μL 40μL(10μM→1μM) 40μL 40μL
      Hg+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 220μL 20μL 40μL 0μL 40μL 40μL
      100μM 40μL 180μL 20μL 40μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 180μL 20μL 40μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 180μL 20μL 40μL 40μL(10μM→1μM) 40μL 40μL
      Cu2+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 260μL 20μL 0μL 0μL 40μL 40μL
      100μM 40μL 220μL 20μL 0μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 220μL 20μL 0μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 220μL 20μL 0μL 40μL(10μM→1μM) 40μL 40μL
      Hg+ buffer DI water Au-NPs Ag+ Mn+ AR H2O2
      target 50mM→5mM 15mM→750μM 100μM→10μM 100μM→10μM 1mM→100μM
      Control 40μL 260μL 20μL 0μL 0μL 40μL 40μL
      100μM 40μL 220μL 20μL 0μL 40μL(1mM→100μM) 40μL 40μL
      10μM 40μL 220μL 20μL 0μL 40μL(100μM→10μM) 40μL 40μL
      1μM 40μL 220μL 20μL 0μL 40μL(10μM→1μM) 40μL 40μL

    4. Result:

      We put all the result into fluorescent reader.

      The horizontal axis:1 means control, 2 means 100μM, 3 means 10μM, 4 means 1μM.And series1 represent no Ag+ adsorb on Au-NPs,series2 stand for the experiment with Ag+ adsorb on Au-NPs which had time mistake(there are one hour shock between adding Mn+ and AR), and the third series is the correct one with Ag+ adsorb on Au-NPs.

    Show More

    Au-NPs affect with different heavy metal (Cu, Pb, Hg,Zn)

    • Researcher: Lin Sheng, Chu Wei-Min
    • Place: NTU Chemistry
    • Date: May 28th, 2015

    Procedure

    1. total:500 μL
    2. material:
      • 3nm Au-NPs 1.5mM,Cu 10mM1mM100μM10μM
      • Cd 10mM1mM100μM10μM,Hg 10mM1mM100μM10μM
      • Zn10mM1mM100μM10μM,pH7 Pb buffer 100mM

    3. Caclulate:

      Cu buffer DI water Au-NPs Mn+
      target 100mM→10mM 1.5mM→3μM 0.1x
      Control 50μL 350μL 100μL 0
      1mM 50μL 300μL 100μL 50μL(10mM→1mM)
      100μM 50μL 300μL 100μL 50μL(1mM→100μM)
      10μM 50μL 300μL 100μL 50μL(100μM→10μM)
      1μM 50μL 300μL 100μL 50μL(10μM→1μM)
      Cd buffer DI water Au-NPs Mn+
      target 100mM→10mM 1.5mM→3μM 0.1x
      Control 50μL 350μL 100μL 0
      1mM 50μL 300μL 100μL 50μL(10mM→1mM)
      100μM 50μL 300μL 100μL 50μL(1mM→100μM)
      10μM 50μL 300μL 100μL 50μL(100μM→10μM)
      1μM 50μL 300μL 100μL 50μL(10μM→1μM)
      Hg buffer DI water Au-NPs Mn+
      target 100mM→10mM 1.5mM→3μM 0.1x
      Control 50μL 350μL 100μL 0
      1mM 50μL 300μL 100μL 50μL(10mM→1mM)
      100μM 50μL 300μL 100μL 50μL(1mM→100μM)
      10μM 50μL 300μL 100μL 50μL(100μM→10μM)
      1μM 50μL 300μL 100μL 50μL(10μM→1μM)
      Zn buffer DI water Au-NPs Mn+
      target 100mM→10mM 1.5mM→3μM 0.1x
      Control 50μL 350μL 100μL 0
      1mM 50μL 300μL 100μL 50μL(10mM→1mM)
      100μM 50μL 300μL 100μL 50μL(1mM→100μM)
      10μM 50μL 300μL 100μL 50μL(100μM→10μM)
      1μM 50μL 300μL 100μL 50μL(10μM→1μM)

    4. Result:

      H1~H4 Hg1mM~1μM

      H4~H8 Cd1mM~1μM

      G1~G4 Cu1mM~1μM

      G5~G8 Zn1mM~1μM

    Show More

    TRANSFORMATION

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: May 28th, 2015

    Materials

    • Resuspended DNA (B0015, E0240, E0840, E0420, E0430, I13001, J23119, B1006)
    • Competent cells (20μl DH5α per transformation)
    • Ice (in ice container)
    • 2ml tube
    • 42℃ water bath
    • Petri dishes with LB agar and appropriate antibiotic
    • Spreader
    • 37℃ incubator
    • SOC Media (180μl per transformation)
    • Pipettman
    • Centrifuge

    Procedure

    1. Start thawing the competent cells on ice.
    2. Add 20 μL of thawed competent cells into pre-chilled 2ml tube, labelled for your control.
    3. Add 1 μL of the DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
    4. Close tubes and incubate the cells on ice for 30 minutes.
    5. Heat shock the cells by immersion in a pre-heated water bath at 42℃ for 60 seconds.
    6. Incubate the cells on ice for 2 minutes for recover.
    7. Add 180 μL of SOC Media(without antibiotic) and then incubate the cells at 37℃ for 1 hour while the tubes are shaking(200~250 rpm).
    8. Centrifuge the cells at 2000 rpm for 2 minutes.
    9. Remove 100 μL of the supernatant by the pipettmen.
    10. For the control, label two petri dishes with LB agar and the appropriate antibiotic(chloramphenicol). Plate 100 μl of the transformation onto the dishes, and spread.
    11. Incubate the plates at 37℃ for 12-16 hours, making sure the agar side of the plate is up.
    12. Store the plate in 4 ℃.
    Show More

    TRANSFORMATION

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: May 21th, 2015

    Materials

    • Resuspended DNA (Resuspend well in 10μl dH20, pipette up and down several times, let sit for a few minutes)
    • Competent cells (100μl DH5α per transformation)
    • Ice (in ice container)
    • 2ml tube (1 per transformation)
    • 42℃ water bath
    • Petri dishes with LB agar and appropriate antibiotic (1 per transformation)
    • Spreader (2 per transformation)
    • 37℃ incubator
    • LB broth (900μl per transformation)
    • Pipettman
    • Centrifuge

    Procedure

    1. Start thawing the competent cells on ice.
    2. Add 100 μL of thawed competent cells into pre-chilled 2ml tube, labelled for your control.
    3. Add 1 μL of the DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
    4. Close tubes and incubate the cells on ice for 30 minutes.
    5. Heat shock the cells by immersion in a pre-heated water bath at 42℃ for 60 seconds.
    6. Incubate the cells on ice for 2 minutes for recover.
    7. Add 900 μL of LB broth(without antibiotic) and thenincubate the cells at 37℃ for 1 hour while the tubes are shaking(200~250 rpm).
    8. Centrifuge the cells at 2000 rpm for 2 minutes.
    9. Remove 800 μL of the supernatant by the pipettmen.
    10. For the control, label two petri dishes with LB agar and the appropriate antibiotic(s). Plate 150 μl of the transformation onto the dishes, and spread.
    11. Incubate the plates at 37℃ for 12-16 hours, making sure the agar side of the plate is up.
    12. After picking colonies, store the plate in 4 ℃.
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    TRANSFORMATION

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: May 7th, 2015

    Materials

    • Resuspended DNA (Resuspend well in 10μl ddH20, pipette up and down several times, let sit for a few minutes)
    • Competent cells (100μl DH5α per transformation)
    • Ice (in ice container)
    • 2ml tube (1 per transformation)
    • 42℃ water bath
    • Petri dishes with LB agar and appropriate antibiotic (1 per transformation)
    • Spreader (1 per transformation)
    • 37℃ incubator
    • LB broth (900μl per transformation)
    • Pipettman
    • Centrifuge

    Procedure

    1. Start thawing the competent cells on ice.
    2. Add 100 μL of thawed competent cells into pre-chilled 2ml tube, labelled for your control.
    3. Add 1 μL of the DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
    4. Close tubes and incubate the cells on ice for 30 minutes.
    5. Heat shock the cells by immersion in a pre-heated water bath at 42℃ for 60 seconds.
    6. Incubate the cells on ice for 2 minutes for recover.
    7. Add 900 μL of LB broth(without antibiotic) and thenincubate the cells at 37℃ for 1 hour while the tubes are shaking(200~250 rpm).
    8. Centrifuge the cells at 2000 rpm for 2 minutes.
    9. Remove 800 μL of the supernatant by the pipettmen.
    10. For the control, label two petri dishes with LB agar and the appropriate antibiotic(s). Plate 50 μl of the transformation onto the dishes, and spread.
    11. Incubate the plates at 37℃ for 12-16 hours, making sure the agar side of the plate is up, and then store the plate in 4 ℃.
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    First-Strand cDNA Synthesis Using M-MLV RT

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: April 30th, 2015

    Material

    1. randon primer 1㎕
    2. 2MUG RNA F10 1.95㎕
    3. 2MUG RNA m1 1.95㎕
    4. 10mM dNTP 1㎕
    5. ddH2O
    6. 5× FSB 4㎕
    7. 0.1M DTT 2㎕
    8. RNase out 1㎕
    9. M-MLV RT 1㎕
    10. eppendorf

    Procedure

    A 20-㎕ reaction volume can be used for 1 ng-5μg of total RNA or 1-500 ng of mRNA.

    1. Add the following components to a nuclease-free microcentrifuge tube:
      • 1㎕ oligo(dT)12-18(500μg/ml), or 50-250 ng random primers, or 2pmole gene -specific primer
      • 1 ng to 5μG total RNA or 1 ng to 500ng of mRNA
      • 1㎕ 10 mM dNTP Mix(10 mM each dATP,dGTP,dCTP and dTTP at neutral pH)
      • Sterile, distilled water to 12㎕
    2. Heat mixture to 65℃ for 5 min an quick chill on ice. Collect the contents of the tube by brief centrifugation and add:
      • 4㎕ 5X First-Strand Buffer
      • 2㎕ 0.1 M DTT
      • 1㎕ RNaseOUTTM Recombinant Ribonuclease Inhibitor (40 units/㎕)

      (Note: When using less than 50 ng of starting RNA, the addition of RNaseOUTTM is essential.)

    3. Mix contents of the tube gently and incubate at 37℃ for 2 min.
    4. Add 1㎕(200 units) of M-MLV RT,a and mix by pipetting gently up and down. If using random primers, incubate tube at 25℃ for 10 min.
    5. Incubate 50 min at 37℃.
    6. Inactivate the reaction by heating at 70℃ for 15 min.

    Result

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    TRANSFORMATION

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: April 30th, 2015

    Materials

    • Resuspended DNA (J04450)
    • Competent cells (100μl DH5α per transformation)
    • Ice (in ice container)
    • 2ml tube
    • 42℃ water bath
    • Petri dishes with LB agar and appropriate antibiotic
    • Spreader
    • 37℃ incubator
    • LB broth (900μl per transformation)
    • Pipettman
    • Centrifuge

    Procedure

    1. Start thawing the competent cells on ice.
    2. Add 100 μL of thawed competent cells into pre-chilled 2ml tube, labelled for your control.
    3. Add 2 μL of the DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
    4. Close tubes and incubate the cells on ice for 30 minutes.
    5. Heat shock the cells by immersion in a pre-heated water bath at 42℃ for 60 seconds.
    6. Incubate the cells on ice for 2 minutes for recover.
    7. Add 900 μL of LB broth(without antibiotic) and thenincubate the cells at 37℃ for 1 hour while the tubes are shaking(200~250 rpm).
    8. Centrifuge the cells at 2000 rpm for 2 minutes.
    9. Remove 800 μL of the supernatant by the pipettmen.
    10. For the control, label two petri dishes with LB agar and the appropriate antibiotic(s). Plate 100 μl and 100 μl of the transformation onto the dishes, and spread.
    11. Incubate the plates at 37℃ for 12-16 hours, making sure the agar side of the plate is up.
    12. After picking colonies, store the plate in 4 ℃.
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    Plasmid Purification & Gel electrophoresis

    • Researcher: Shen Yu-Chun, Chen Sheng-Yuan
    • Place: NTUCM
    • Date: April 30th, 2015

    Material:

    1. Resuspension Buffer S1
    2. Lysis Buffer S2
    3. Neutralization Buffer S3
    4. Equilibration Buffer N2
    5. Wash Buffer N3

    6. Elution Buffer N5

    7. Overnight bacteria
    8. Room-temperature isopropanol
    9. 1% agarose
    10. TE buffer (using at Gel electrophoresis)

    Protocol:(Plasmid Purification)

    1. Harvest bacteria from an LB culture by centrifugation at 6000 rpm for 10 mins at 4℃

    2. Resuspend the pellet of bacterial cells in Buffer S1.

    3. Add Buffer S2 the suspension .Mix by inverting the tube for 6~8 times. Incubate the mixture at 18~25℃ for 2~3 mins.
    4. Add Buffer S3 (pre-cooled at 4℃ ) to the suspension. Immediately mix the lysate by inverting the flask 6~8 times.
    5. Equilibrate a Column with Buffer N2

    6. Place the folded filter in a funnel of appropriate size. Wet the filter with a few drop of Buffer N2 and load the bacterial lysate onto the wet filter.

    7. Load the cleared lysate from 6 onto the column. Allow the column to empty by gravity flow.
    8. Wash the column with Buffer N3.
    9. Elute the plasmid DNA with Buffer N5.
    10. Add room-temperature isopropanol to precipitate the eluted plasmid DNA. Mix carefully and centrifuge at 12000 g for 30 mins at 4℃. Carefully discard the supertant.
    11. Add room-temperature 70% ethanol to the pellet. Vortex briefly and centrifuge at 12000 g for 10 min at 18~25℃.
    12. Carefully remove ethantol from the tube with a pipette tip. Allow the pellet to dry at 18~25℃ no iess than the indicated time.
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    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: April 30th, 2015

    Procedure

    Transform T7promoter-RBS-QsrR BBa_K1092001

    Show More

    First-Strand cDNA Synthesis Using M-MLV RT

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: April 30th, 2015

    Preparing Samples

    Homogenizing samples

    • Determine your sample type, and perform homogenization at room temperature according to the table below. The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization. Be sure to use the indicated amount of TRIzol Reagent, because an insufficient volume can result in DNA contamination of isolated RNA.
    Sample Type Action
    Tissues
    1. Add 1mL TRIzol Reagent per 50-100 mg of tissue sample.
    2. Homogenize sample using a glass-Teflon or power homogenizer.
    • Note: Process or freeze tissue samples immediately upon collection.

    RNA Isolation Procedurec

    Always use the appropriate precautions to avoid RNase contamination when preparing and handling RNA.

    RNA precipitation

    1. (Optional) When precipitating RNA from small sample quantities (<106 cells or < 10 mg tissue), add 5-10μg of RNase-free glycogen as a carrier to the aqueous phase.
      • Note: Glycogen is co-precipitated with the RNA,but does not inhibit first-strand synthesis at concentrations ≤4mg/mL,and does not inhibit PCR.
    2. Add 0.5mL of 100% isopropanol to the aqueous phase, per 1mL of TRIzol Reagent used for homogenization.
    3. Incubate at room temperature for 10 minutes.
    4. Centrifuge at 12,000 × g for 10 minutes at 4℃.
      • Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube.
    5. Proceed to RNA wash.

    Proceed to RNA wash.

    1. Remove the supernatant from the tube,leaving only the RNA pellet.
    2. Wash the pellet,with 1mL of 75% ethanol per 1mL of TRIzol Reagent used in the initial homogenization.
      • Note: The RNA can be stored in 75% ethanol at least 1 year at-20℃, or at least 1 week at 4℃.
    3. Vortex the sample briefly,then centrifuge the tube at 7500 × g for 5 minutes at 4℃.Discard the wash.
    4. Vacuum or air dry the RNA pellet for 5-10 minutes. Do not dry the pellet by vacuum centrifuge.
        Note: Do not allow the RNA to dry completely,because the pellet can lose solubility.Partially dissolved RNA samples have an A260/280 ratio<1.6.
    5. Proceed to RNA resuspension.

    RNA resuspension

    1. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution(20-50㎕) by passing the solution up and down several times through a pipette tip.
      • Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions.
    2. Incubate in a water bath or heat block set at 55-60℃ for 10-15 minutes.
    3. Proceed to downstream application, or store at -70℃.
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    Transformation

    • Researcher: Shen Yu-Chun, Chen Sheng-Yuan
    • Place: NTUCM
    • Date: April 13th, 2015

    Material:

    1. E.coli DH5α (ps: E.coli should be kept on the ice at all time)
    2. Plasmid pBR322(Ampicillin-resistant)
    3. LB broth (10 g /L Tryptone, 5 g/L Yeast extract, 5 g/L NaCl )
    4. LB plate(LB broth + 1% agar)
    5. Antibiotics : Ampicillin(100μg/mL)

    Protocol:

    1. Add 5μL of DNA into 100μL of E.coli and mix by tapping the tube. (ps: the quantity of DNA is dependent on bacterial competency)

    2. Put the tube on the ice for 20~30mins.

    3. Remove from the ice and put the tube to the 42℃ hot bath for 45 sec.

    4. After heat shock, put the tube into the ice IMMEDIATELY for 2 mins.

    5. Add 500 μL of LB broth into the tube and incubate for 30~60 mins at 37℃
    6. Take out the tube and put into 6000 rpm centrifuge for 5 mins
    7. Remove supertant and remain about 100μL of LB to resuspend
    8. Spread onto LB plate containing the appropriate antibiotic

    9. Incubate overnight for 12~16 hrs at 37℃ and you will see so many colonies on the LB plate!!!(A colony is a white dot on the picture below)

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    Plasmid Extraction

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: April 2nd, 2015

    Procedure

    1. Harvesting

      2. Transfer 1.5ml of cultured bacterial cells to a 1.5ml microcentrifuge tube. Centrifuge at 13000 x g for 1 minute at room temperature.

    2. Resuspension

      3. Add 200μl of PD1 Buffer to a new 1.5ml microcentrifuge tube. Add 2μl of TrueBlue Lysis Buffer to the same 1.5ml microcentrifuge tube then mix by shaking gently.

    3. Cell Lysis

      4. Add 200μl of PD2 to the resuspended sample then mix genlly by inverting the tube 10 times. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.

    4. Neutralization

      5. Add 300 μl of PD3 Buffer then mix immediately by inverting the tube 10 times. Centrifuge at 13000 x g for 3 minutes at room temperature.

    5. DNA Binding

      6. Transfer all of the supernatant to thePDH Columg. Centrifuge at 13000 x g for 30 seconds at room temperature then discard th flow-through. Place the PDH Column back in the 2 ml Collection Tube.

    6. Wash

    For Improved Downstream Sequencing Reactions

    Add 400 μl of W1 Buffer into the PDH Column. Centrifuge at 13000 x g for 30 seconds. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube. Proceed with Wash Buffer addition.

    For Standard Plasmid DNA Purification

    Add 600 μl of Wash Buffer into the PDH column. Centrifuge at 13000 x g for 30 seconds at room temperature. Discard the flow through then place the PDH Column back in the 2 ml Collection Tube. Centrifuge at 13000 x g for 3 minutes at room temperature to dry the column matrix.

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    Colony PCR

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: April 2nd, 2015
    1. PCR mixer
      2. 10X PCR Buffer,-Mg to 100μl
      50mM MgCl2 10μl
      10mM dNTP Mix 3μl
      Taq DNA Polymerase(5U/μl) 0.4μl
      10μM forward primer 5μl
      10μM reverse primer 5μl

      ∗Mix briefly centrifuge the components.

    2. Put the mix into each of ten tubes. Eight of them are colonies, and the others are positive and negative control.
    3. Put all tubes into the PCR machine and set up like below:
    4. Step Temperature(℃) Time
      Initial Denaturation 95 5min
      30 PCR Cycles Denature 94 15sec
      Anneal 55 15sec
      Extend 72 30sec
      Final Extension 72 7min
      Hold 25 indefinitely
    5. When it finished, we run gel.
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    Transformation

    • Researcher: Chang Ko-Yu, Chu Yi-Chia
    • Place: Academia Sinica
    • Date: March 26th, 2015

    Material needed

    1. 70 % ethanol
    2. Bba_J23102 (2λ)
    3. Dn5α (20λ)
    4. soc (200λ)
    5. Peni 50 mg/ml
    6. chrlo 34mg/ml
    7. ministart
    8. dish x10
    9. pipetman,tip
    10. eppendorf
    1. Add 2λ plasmid and 20λ E-coli into PCR tube in the ice bucket.
    2. Place the tubes on ice for 30min
    3. Heatshock 42℃ for 45secs
    4. After Heatshock, put into the ice bucket for 30 min.
    5. Add soc 200λ to repair the cell wall Culture in the 37℃ incubator for 1.5 hr

    Finish smear dish in hood.

    Result

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    Transform

    • Researcher: Zhao Ming-Cheng,Su Chin-Fong
    • Place: NTU
    • Date: March 26th, 2015

    Procedure

    Step1：Spread some culture media on the petri dish and heated in 37℃ for 20min in the oven.

    STEP2：Take the plastid and bacterium out of the refrigerator(-80℃) and heated in 37℃ for 3min in the oven.

    STEP3：Take the plastid and bacterium out and put it into the centrifuge.

    STEP4：Use a L-type glass rod and spread the plastid and bacterium on the petri dish tell it become gluey.

    STEP5：Cover up the petri dish and wait for about 16 to 24 hours to reform.

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    Ligation

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: March 26th, 2015

    1. To an autoclaved, 1.5ml microcentrifuge tube, add the following:
      For Cohesive Ends
      5X Ligase Reaction 4 μl
      Vector D 3 to 30 fmol
      Insert D 9 to 90 fmol
      T4 DNA Ligase ( 1 unit (in 1 μl)
      Autoclaved distilled to 20 μl
    2. Mix gently. Centrifuge briefly the contents to the bottom of the tube.
    3. Incubate at room temperature for 5 min.
    4. Use 2 μl of the ligation reaction to transform 100 μl of competent cells.
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    3/19/2015

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU
    • Date: Match 19th, 2015

    Add restriction enzyme (EcoRI / SpeI)

    Total:40ul

    Plasmid DNA:500ng (77.7ng/ul, 500/77.7≒6.5ul)

    　 　 　 　 　 　 　
    ddH2O 22.5ul
    10Xbfr(EcoRI) 4ul
    10XBSA 4ul
    Plasmid DNA 6.5ul
    EcoRI 1.5ul
    SpeI 1.5ul
    Total 40ul
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    Gel Extraction

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: March 19th, 2015
    1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
    2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel~100 μl).The maximum amount of gel per spin column is 400 mg.For>2% agarose gels,add 6 volumes Buffer QG.
    3. Incubate at 50℃ for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
    4. Add 1 gel volume isopropanol to the sample and mix.
    5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of&62;800μl, load and spin/apply vacuum again.
    6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
    7. To wash, add 750μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, bluntended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
    8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
    9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
    10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
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    High Copy Number Protocol

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: TMU lab
    • Date: March 18th, 2015

    Harvesting

    1. Transfer 1.5ml of bacterial culture to a microcentrifuge tube. Centrifuge for 2 min at 6,000 rpm in a microcentrifuge and discard the supernatant.

      Resuspension

    2. Add 200μl of PS1 Buffer(RNaseA added)to the tube and resuspend the cell pellet by vortexing or pipetting.

      Lysis

    3. Add 200μl of PS2 Buffer and mix gently by inverting the tube 10 times. Do not vortex, avoid shearing genomic DNA. Stand for 3 minutes at room temperature until lysate clears.

      Neutralization

    4. Add 300μl of PS3 Buffer and mix immediately by inverting the tube 10 times. Do not vortex. Until the precipitate formation, shake a few times to broken it. Incubate at room temperature for 5 minutes to digest RNA. Centrifuge for 5 minutes at 13,000 rpm.

      DNA Binding

    5. Place a PS Column in a Collection tube. Apply the clear lysate (supernatant) from Step 4 to the PS Column. Centrifuge at 13,000 rpm for 30 seconds.

    6. Discard the flow-through and place the PS Column back into the Collection Tube.

      Wash

    7. Add 400μl of W1 Buffer in the PS Column. Centrifuge at 13,000 rpm for 30 seconds.
    8. Discard the flow-through and place the PS Column back into the Collection Tube.
    9. Add 600μl of W2 Buffer(ethanol added) in the PS Column. Centrifuge at 13,000 rpm for 30.

    10. Discard the flow-through and place the PS Column back into the Collection Tube. Centrifuge again for 3 minutes at 13,000 rpm to dry the Column matrix.

      DNA Elution

    11. Transfer dried PS Column to a clean microcentrifuge tube. Add 50μl of distilled water into the center of the column matrix. Stand for 2 minutes until distilled water is absorbed by the matrix. Centrifuge for 2 minutes at 13,000 rpm to elute purified DNA.
    12. If would like more production, repeat this elution step again.
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    TRANSFORMATION

    • Researcher: Han Yun-An, Chang Yu-Ting, Lee Chang-Lung
    • Place: Taipei Medical University
    • Date: March 12th, 2015

    Materials

    • Resuspended DNA (Resuspend well in 10μl dH20, pipette up and down several times, let sit for a few minutes)
    • Competent cells (100μl DH5α per transformation)
    • Ice (in ice container)
    • 2ml tube (1 per transformation)
    • 42℃ water bath
    • Petri dishes with LB agar and appropriate antibiotic (2 per transformation)
    • Spreader (2 per transformation)
    • 37℃ incubator
    • 10pg/μl RFP Control (pSB1C3 w/ BBa_J23102)
    • LB broth (900μl per transformation)
    • Pipettman
    • Centrifuge

    Procedure

    1. Start thawing the competent cells on ice.
    2. Add 100 μL of thawed competent cells into pre-chilled 2ml tube, labelled for your control.
    3. Add 1 μL of the DNA with RFP Control to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
    4. Close tubes and incubate the cells on ice for 30 minutes.
    5. Heat shock the cells by immersion in a pre-heated water bath at 42℃ for 60 seconds.
    6. Incubate the cells on ice for 3 minutes for recover.
    7. Add 900 μL of LB broth(without antibiotic) and thenincubate the cells at 37℃ for 1 hour while the tubes are shaking(200~250 rpm).
    8. Centrifuge the cells at 2000 rpm for 2 minutes.
    9. Remove 800 μL of the supernatant by the pipettmen.
    10. For the control, label two petri dishes with LB agar and the appropriate antibiotic(s). Plate 50 μl and 150 μl of the transformation onto the dishes, and spread.
    11. Incubate the plates at 37℃ for 12-16 hours, making sure the agar side of the plate is up.
    12. After picking colonies, store the plate in 4 ℃.
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