Difference between revisions of "Team:HSNU-TAIPEI/labnotes"

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Revision as of 09:35, 13 September 2015

Lab Notes

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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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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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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Transformation

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

We transform J22106 this time.

Show More

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
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

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.

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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

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

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.

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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.

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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

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8/4/2015 Cadmium

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

We successfully draw out the plasmid.

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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.

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Transformation

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

We transform J23119 this time.

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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.

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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.

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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.

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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.

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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.

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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}

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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.

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7/13/2015 Cadmium

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

We get “k89608” part and transform it.

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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.

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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.
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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.

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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.

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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.

Show More

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℃.
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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.

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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.

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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

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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 ℃.
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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-5MUG 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 5MUG 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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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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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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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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