1)  Experimental notes and process

l  LB media

- Objective: Culture and Amplify E. coli

- Concepts:

1.  tryptophan and yeast extract provide carbon and nitrogen needed by E. coli.

2.  NaCl helps maintain the osmotic pressure.

- Materials: 

Name

Dosage

Tryptone

10 g/L

Yeast extract

5 g/L

NaCl

10 g/L

  Note: solid: +3 g agar powder

- Methods:

1.  Use an electronic balance to weigh the required reagents separately.

2.  Mix tryptone, yeast extract, NaCl powder, agar powder, and water in a conical .  Shake the container until the solute dissolves. Adjust pH to 7.0 with 5mol/LNaOH. Adjust volume to 1L with deionized water.

3.  Use millipore filter membrane to seal the conical flask.

4.  Place a piece of autoclave indicator tape on the conical flask .

5.  Steam sterilization was performed at 121 ° C for 21min.

- Results: 

1.  Autoclave indicator tape turns black

2.  The solution shows a yellow color

l  PCR

- Objective: Amplify a specific DNA fragment

- Concepts: At 95 , the DNA helix unwinds transforme d  from double to single. When held at 72  for 10 min, all the single chains will become double. During the cycle, the structure of DNA transformed repeatedly between single and double strands.

- Materials: 

Mix: Mg 2+ , DNA polymerase, dNTP, ddH 2 O

     

Amplifying the target gene of plasmid 1 (zliE-zliS )    

Name

Dosage μL

Mix

10

Primer: zliE-F1

1

Primer: zliS-R1

1

DNA template: zm4

1

dd H2O

7

Total

20

 

     Amplifying the target gene of plasmid 2 (SacB)

Name

Dosage μL

Mix

10

Primer: SacB-F1

1

Primer: SacB-R1

1

DNA template: zm4

1

ddH2O

7

Total

20

- Methods: 

1.  Mix some of the reagents mentioned above in a centrifuge tube separately (group 1: zliE F1 + zliS R1 + ddH2O + mix + template zm4; group 2: SacB F1 + SacB R1 + ddH2O + mix + template zm4 ), except zm4, ( all tubes should be placed in crushed ice, otherwise most enzyme will become inactive, such as DNA polymerase )

2.  Vortex and centrifuge (to avoid the occurrence of solution hanging on the wall of the tube and Mix well )

3.  Place in PCR machine

4.  Turn on the machine

5.  Click “file” on the screen

6.  Click “program

7.  Click “primmer start”

PCR machine program settings:

steps

temperature

time

1

95

3 min

2

95

30 sec

3

55

30 sec

4

72

1min

5

72

10 min

6

4

forever

PS: Repeat 2-4 30 cycles

- Result: Achieving DNA amplification

l  Gel electrophoresis 

- Objective: To check whether the DNA sample contains the correct DNA segments/fragments we need

- Concepts: The gel we constructed has a porous structure, which provides small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the correct DNA fragments we need according to the marker.  

- Materials:

Name

Dosage

TAE buffer

100 mL

Agarose

1%-->1 g

YeaRed nucleic acid gel stain

1/10 thousand-->10 μL

- Methods: (note: the containers used in this process should be marked with the words “TAE special”)

1.  Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a erlenmeyer flask

2.  Place the solution in the microwave on medium-high heat for 2 min repeatedly until the solution boils dissolve ( to ensure uniform mixing)

3.  Fix comb in the mold (thick: 50 mL; thin: 20 mL )

4.  Pour the agarose gel slowly into the mold, taking care not to create bubbles Wait until the agarose gel setsRemove the comb

5.  Pour TAE buffer solution into the tank

6.  Use the pipette to add the four DNA samples made in the step “PCR” and marker to the corresponding grooves respectively.

7.  Put the lid on and power on (180V, 20 min)

8.  (After 20 min) put the gel under a gel imager to document bands

9.  Software: Genosens

10.  Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” + “collect” + “save” + name the graph (date + reagent name)

- Result: group 2 SacB DNA successfully separated

l  DNA extraction

- Objective: separate the target DNA fragments from the gel and detect the concentration of DNA

- Concepts: binding, washing and eluting from a silica column

- Materials:

 Silica column

Name

Dosage ( μL)

Buffer 2

300

Wash solution

500

Elution buffer

40

- Methods: (note: protein: 280 nm; DNA: 260 nm )

 

1.  Cut out the gel with target DNA

2.  Mix the gel that contains DNA with 300µL Buffer 2 in a tube

3.  Place the tube in a water bath at 65˚C for 5-10 min

4.  Centrifuge (8,000 × g ) for 30 s and pour off the underlying liquid ( liquid in the transparent tube )

5.  Add 500 µL wash solution

6.  Centrifuge (9,000 × g ) for 30 s and pour off the underlying liquid ( liquid in the transparent tube )

7.  R epeat 5 and 6

8.  Centrifuge (9,000 × g ) for 30 s and pour off the underlying liquid ( liquid in the transparent tube )

9.  Centrifuge (9,000 × g ) for 1 min

10.  Place the solution (with the internal yellow tube ) into two new 1.5 mL tubes

11.  Add 40 µL elution buffer into tube

12.  Wait for 1 min

13.  Centrifuge (9,000 × g ) for 1 min

14.  Detect DNA concentration  

15.  Click “nucleic acid” on the screen

16.  Add 1 µL elution buffer into the “small hole” on the instrument

17.  Click “empty test” + “zero”

18.  Dropping 1 µL DNA sample to detector

19.  Click “sample testing”

(note: normally: A260/A280: 1.8-2.1 )

- Results: 

1.  Concentration: 0.9ng/µL

2.  A260/A280: 2.25

 (reasons: RNA/DNA remains a lot of fragments; if the figure for A280 is higher than the normal range ( 1.8-2.1 ): some proteins have not been cleaned up )

l  Inverse PCR

- Objective: amplify a specific DNA fragment

- Concepts: Because there is no suitable enzyme cutting site on this structure, or there is a terminator blocking the normal direction, it is necessary to change the direction for PCR. The purpose of inverse PCR is to amplify DNA flanking a known sequence, that is to say, this reaction system synthesizes DNA not between a pair of primers but outside the primers.

- Materials:

Name

Dosage (µL)

Template: plasmid 4

1

Primer: zliE-F1

1

Primer: SacB-F4-avr

1

mix

20

ddH 2 O

17

Total

40

Mix: Mg 2+ , DNA polymerase, dNTP (SacB fragment ), ddH 2 O

- Methods:

1.  Mix all reagrens mentioned above ( add them in order from largest to smallest )

2.  Place the tube inside the centrifuge machine

3.  Take out the tube and place it in a PCR machine

4.  Turn on the machine

5.  Click “file” on the screen

6.  Click “program

7.  Click “primmer start”

steps

temperature

time

1

95

3 min

2

95

30 s

3

55

30 s

4

72

5 min

5

72

10 min

6

4

forever

PS: Repeat 2-4 30 cycles

- Result: Achieving DNA amplification

 

1.  Continue to complete the remaining reverse PCR on the 21st

2.  Complete the unsuccessful process on the 21st: PCR: primmer: zliE F1 + zliS R1

3.  Restriction digestion of empty plasmid and target fragment

4.  Enzyme digestion to verify EV (empty vector )

5.  Recycle DNA

6.  Enzyme-linked plasmid fragments and target fragment

 

l  PCR (single--->double; Annealed PCR )

- Objective: Amplify the single-stranded gene (plac and puv) purchased from the company into a double-stranded structure (amplify plac and puv), and use T4 ligase to help connect them. And finally provide materials for the subsequent process.

- Concepts: When a single-stranded gene is heated and annealed, it is amplified and double-stranded.

- Materials:  

Name

Dosage ( μL)  

T4 ligase buffer

2

Primer 1

5

Primer 2

5

ddH 2 O

8

Total

20

- Methods:

1.  Mix all the reagents mentioned above in a centrifuge tube

2.  Place the tube inside the centrifuge (to avoid the occurrence of solution hanging on the wall of the tube and mix well )

3.  Take out the tube and place the tube inside a PCR machine

4.  Turn on the machine

5.  Click “file” on the screen

6.  Click “program

7.  Click “primmer start”

steps

temperature

time

1

95

5 min

2

80

1 min

3

70

1 min

4

60

1 min

5

50

1 min

6

40

1 min

- Results: Successfully changed the single-stranded structure into double-stranded, and amplified.

l  Digestion (empty vector: pUC 19 )

- Objective: The cohesive ends on the fragments are cut out with enzymes to facilitate fusion with other fragments later. Cut out a sticky end of pUC 19 (EV ) for building up plasmid 1 in later process.

- Concepts: Cut out identical sticky ends to aid binding

- Materials:

 

Name

Dosage (µL)

pUC 19 (EV)

30

Enzyme 1: Sal I

1

Enzyme 2: pst I

1

rCutSmart

5

dd H 2 O

13

Total

50

- Methods:

1.  Mix all reagents  mentioned above in a tube

2.  Place the test tube in a water bath at 37˚C for 30 min (This is the optimum environment for enzyme growth )

- Results: Digestion was successfully completed and the cohesive ends were cut out.

l  Digestion (annealed PCR product ( plac/puv) and IPCR: plasmid 4 )

- Objective: The cohesive ends on the fragments are cut openby  enzymes to facilitate fusion with other fragments later.

- Concepts: The cleaved fragments have identical cohesive ends to facilitate later joining. The gene fragment made before has no suitable restriction site, and the added primers provide it with restriction sites. The annealed PCR product is also equivalent to providing two primers, so that the target gene can have a site.

- Materials:

Name

Dosage (µL)

IPCR product--plasmid 4/annealed PCR product--plac & puv

10

Enzyme 1: Avr II

1

Enzyme 2: Sal I

0.5

rCutSmart

2

ddH 2 0

6

- Methods:

1.  Mix reagents  mentioned above in two tubes separately

a)  Group 1: annealed PCR product—plac + both two types of enzyme + rCutSmart + dd H 2 O

b)  Group 2: annealed PCR product—puv + both two types of enzyme + rCutSmart + dd H 2 O

2.  Group 3: IPCR product--plasmid 4+both two types of enzyme + rCutSmart + dd H 2 O  Place the tubes in a water bath at 37 ˚C for 30 min

- Results: Digestion was successfully completed and the cohesive ends were cut out.

l  E nzyme-link (mix digested plac for plasmid 5 or digested puv for plasmid 6 with annealed PCR product respectively )

- Objective: Ligate and fuse the previously cut plasmid and target gene with the same cohesive ends.

- Concepts: Ligase helps gene fragments join and fuse.

- Materials:

 

Name

Dosage (µL)

T4 ligase

1

T4 ligase buffer

2

Digested plac for plasmid 5

 Digested puv for plasmid 6

 IPCR product: plasmid 4

- Methods:

1.  Mix digested IPCR plac, IPCR product--plasmid 4, T4 ligase, and T4 ligase buffer in a tube

2.  Mix digested IPCR puv, IPCR product--plasmid 4, T4 ligase, T4 ligase buffer in a tube

- Results: Fuse the target gene and plasmid together. Build up plasmid 5 and plasmid 6.

 

Day 3

1.  PCR amplify RFP (outline: prepare for RFP; plasmid 2--->plasmid 3)

2. Homologous recombination (homology arm )

3. Transform into DH5 α  E.coli and grow overnight.

 

l  PCR (RFP )

- Objective: Amplify RFP to 687bp, prepare reagents for building up plasmid 3

- Concepts: At 95 , the DNA helix unwinds transforme d  from double to single. When held at 72  for 10 min, all the single chains will become double. During the cycle, the structure of DNA transformed repeatedly between single and double strands.

- Materials: 

Name

Dosage μL

Mix

10

Primer: RFP-F2

1

Primer: TrrnB-R1

1

DNA template: puc57-RFP

1

ddH2O

7

Total

20

 Mix: Mg 2+ , dNTP, DNA polymerase

- Methods:

1.  Mix both of the two primers, DNA template, ddH2O, and the mix in a centrifuge tube (follow the order from large quantity to small quantity )

2.  vortex and centrifuge (to avoid the occurrence of solution hanging on the wall of the tube and mix well )

3.  Place the tube inside the PCR machine

4.  Place in PCR machine

5.  Turn on the machine

6.  Click “file” on the screen

7.  Click “program

8.  Click “primmer start”

steps

temperature

time

1

95

3 min

2

95

30 sec

3

55

30 sec

4

72

30 sec

5

72

10 min

6

4

forever

PS: Repeat 2-4 30 cycle s

- Result: amplify RFP to about 687 bp

l  Homologous recombination ( RFP and IPCR--->plasmid 3 )

- Objective: Fusion of plasmid 2 (IPCR product) and target gene RFP. Build up plasmid 3.  

- Concepts: Because there is no suitable enzyme cutting site, a homology arm is used to forcibly connect the two gene fragments. Find Homology arms: Compare the sequence and base pairs of two DNA fragments. Homologous recombination refers to the exchange of time that occurs between two homologous chromosomes on a chromosome. The gene fragments are first cut with double-strand breakase, the homologous arms are reconnected with homologous recombinase, and finally the chromosome is connected with DNA ligase.

- Materials: 

 

Name

Dosage (µL)

Template: IPCR product: plasmid 2

2

Target gene: RFP

1

Mix

5

ddH 2 O

2

Total

10

6000bp*0.02=120 ---------- 2 µL template: IPCR product: plasmid 2

687bp*0.04=28 ---------- 1 µL target gene: RFP

     Mix: dNTP, Mg 2+ , DNA polymerase

- Methods:

1.  Mix the template, the target gene, ddH2O, and the mix in a tube.

2.  Put the tube into a water bath machine at 37˚C for 5 min

3.  Place the tube on crashed ice for 3 min

- Result: Embedding of target gene and template gene; completely build up plasmid 3.

l  Transformation (Insertion of three plasmids into E. coli )

- Objective: Let E. coli help increase the number of plasmid 3, 5 and 6

- Concepts: E. coli can keep growing to continuously replicate our implanted genes.

- Materials:

Name

Dosage (µL)

Plasmid 5/plasmid 6/RFP after homologous recombination--plasmid 3

20

Solution that contains E.coli 

- Methods:

1.  Mix the reagents separately (group 1: plasmid 3 + solution that contains E.coli; group 2: plasmid 5 + solution that contains E.coli; group 3: plasmid 6 + solution that contains E.coli )

2.  Place them into crashed ice for 30 min

3.  Water bath Heat shock at 42 ˚C for 45 s ( let the E.coli  devour the three plasmids we want to implant faster and better; only 45 s, because if it take longer than that, the E.coli  will die due to high temperatur e  )

4.  Place them into crashed ice again, for only 2-3 min

- Result: all plasmid 3, plasmid 5, and plasmid 6 were implanted in E.coli respectively.

l  Culture E.coli with three different plasmids

- Objective: we need to get E.coli to help expand the production of plasmid 5, plasmid 6, and plasmid 3.

- Concepts: the LB media provides a suitable environment which is beneficial for E.coli to grow. (LB media: tryptophan and yeast extract provide carbon and nitrogen needed by E. coli.  NaCl helps maintain the osmotic pressure)

- Materials:

 Part 1:

Name

Dosage (µL)

LB media ( solution )

900

Mixture 1

/

Mixture 2

/

Mixture 3

/

Ps: Mixture 1: plasmid 3 + E.coli

 Mixture 2: plasmid 5 + E.coli

 Mixture 3: plasmid 6 + E.coli

Name

Dosage (µL)

Prepared solid AMP + LB media

/

Mixture 1

10

Mixture 2

10

Mixture 3

10

Ps: Mixture 1: liquid LB media+E.coli+plasmid 3

  Mixture 2: liquid LB media+E.coli+plasmid 5

      Mixture 3: liquid LB media+E.coli+plasmid 6

- Methods:

 Part 1:

1.  Spray the gloves with alcohol and check clothing on arms, hands, etc., before you put your hands in the tank (to make sure no skin is exposed to the air )

2.  Light the alcohol lamp

3.  Mix 900 µL LB media and E.coli  with plasmid 3 in a new tube.

4.  Mix 900 µL LB media and E.coli  with plasmid 5 in a new tube.

5.  Mix 900 µL LB media and E.coli with plasmid 6 in a new tube.

(notion: things are all made inside the sterilizing tank; from 4-6, all process should be operated near the alcohol lamp )

6.  Place them inside a machine, keep constant shaking for 1 h

7.  Transfer the tubes to the sterilizing tank

Part 2:

1.  Spray the gloves with alcohol and check clothing on arms, hands, etc., before you put your hands in the tank (to make sure no skin is exposed to the air )

2.  Light the alcohol lamp

3.  Peel off the membrane on the side containing the solid LB media petri dish

4.  Transfer 10 µL mixture (liquid LB media + E.coli  + plasmid 3) onto a prepared solid LB + AMP media

5.  Transfer 10 µL mixture (liquid LB media + E.coli + plasmid 5) onto a solid LB + AMP media

6.  Transfer 10 µL mixture (liquid LB media + E.coli + plasmid 6) onto a solid LB + AMP media

7.  Use a disposable scrapeer to evenly apply the mixture to the solid LB + AMP media’s surface respectively

8.  Seal the sides of the petri dish

9.  Turn the petri dish upside down (place the liquid coated side down ), and place it in an incubator, waiting for E.coli to grow.

- Result: wait for tomorrows observation.

 

 

l  PCR (E.coli with plasmid 3/plasmid 1 ( RFP without its own promoter ) )

- Objective: amplify the E.coli with plasmid 1 and plasmid 3

- Concepts: At 95 , the DNA helix unwinds transforme d  from double to single. When held at 72  for 10 min, all the single chains will become double. During the cycle, the structure of DNA transformed repeatedly between single and double strands.

- Material:

 Group 1 ( E.coli with plasmid 1 )

Name

Dosage (µL)

Mix

10

Primer 1: TrrnB-R1

1

Primer 2: RPT-F2

1

Template: a single strain of E.coli with plasmid 1

/

ddH2O

8

Total

20

 Group 1 ( E.coli with plasmid 1 )

Name

Dosage (µL)

Mix

10

Primer 1: TSacB-F1

1

Primer 2: zli-SacB-BkR1

1

Template: a single strain of E.coli with plasmid 3

/

ddH 2 O

8

Total

20

- Methods:

1.  Mix ddH 2 0, mix, and two primers in a PCR tube

2.  Select a single strain of E.coli

a)  Spray the gloves with alcohol and check clothing on arms, hands, etc., before you put your hands in the tank (to make sure no skin is exposed to the air )

b)  Light the alcohol lamp

c)  Peel off the membrane on the side containing the solid LB media petri dish (both of the small one that contains E.coli with plasmid 1/plasmid 3 ) and the larger one that only contains solid LB media )

d)  Use the pipette to pick up a single strain with the tip of the pipette

e)  Transfer the single strain on to the surface of solid LB media in a larger petri dish

f)  Draw a line on it with the tip of the pipette (Draw a marker next to the first line )

g)  Continue to draw lines after the first line, about 7-8 or so (withou t  crossing each other; remember to mark the corresponding serial number on the outside of the Petri dish with a marker )

h)  Put the tip of the pipette back into the PCR tube, hold the tip with your hand, and stir it

3.  Vortex and centrifuge (to avoid the occurrence of solution hanging on the wall of the tube )

4.  Place in PCR machine

5.  Turn on the machine

6.  Click “file” on the screen

7.  Click “program

8.  Click “primmer start”

steps

temperature

time

1

95

3 min

2

95

30 s

3

55

30 s

4

72

1 min

5

72

10 min

6

4

forever

PS: Repeat 2-4 30 times

- Result: Achieving the amplification of E.coli that contains plasmid 1/plasmid 3

l  AMP solid medium

- Objective: to kill most of the E.coli that is not implanted with plasmad1 or plasmid 3

- Concepts: The implanted E. coli is resistant to ammonia, so only the E. coli implanted with plasmid 1 and plasmid 3 can survive in the presence of AMP.

- Materials:

Name

Dosage

Stock solution (AMP)

100 mg/L

LB media

35 mL

AMP solution

40 µL

Our target concentration: 100 µg/L

100mg/L stock solution (AMP)

 To achieve the target concentration: 1ml LB+1µl stock solution (AMP )

 Total (LB+AMP ): 3 m L /plate*20 plate=60 m L  solid LB

 70 m L -->70 µL AMP-->80µL AMP

 50 mL centrifuge tube+35mL LB+40µL AMP

- Methods:

10.  Spray the gloves with alcohol and check clothing on arms, hands, etc., before you put your hands in the tank (to make sure no skin is exposed to the air )

11.  Light the alcohol lamp

12.  Mix 35 mL LB and 40 µL AMP solution in a tube

13.  Transfer 3 mL of the mixture into a new smaller tube

14.  And then let the bacterial liquid evenly distribute on the surface of the culture medium

15.  Seal the side of the petri dish

16.  Wait for solidification

- Result: solidified

l  Gel electrophoresis 

- Objective: To check whether the grown E.coli contains the correct DNA segments/fragments we need

- Concepts: The gel we constructed has a porous structure, which provides small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the correct DNA fragments we need according to the marker.

- Materials:

Name

Dosage

TAE buffer

100 mL

Agarose

1%-->1 g

YeaRed nucleic acid gel stain

1/10 thousand-->10 μL

- Methods: (note: the containers used in this process should be marked with the words “TAE special”)

1.  Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a erlenmeyer flask

2.  Place the solution in the microwave on medium-high heat for 2 min repeatedly until the solution boils (to ensure uniform mixing)

3.  Fix comb in the mold (thick: 50 mL; thin: 20 mL)

4.  Pour the gel

5.  Wait until it turns into a solid gel

6.  Remove the comb

7.  Pour TAE solution into the tank

8.  Use the pipette to add the four reagents made in the step “PCR” and marker to the corresponding grooves respectively.

9.  Put the lid on and power on (180V, 20 min)

10.  (After 20 min) put the gel under a gel imager to document bands

11.  Software: Genosens

12.  Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” + “collect” + “save” + name the graph (date + reagent name)

- Results: 

1.  Plasmid 3: contains the correct DNA segments/fragments except 3-2-6, 3-2-7, 3-2-8

   Reasons (might be ):

a)  Monoclonal strains were not picked clean

b)  Some reagents are not sucked clean

c)  There is liquid hanging on the tube when transferring liquid, or gloves pick up some liquid when opening the test tube

2.  Plasmid 1: all failed

   Reason (might be ):

a)  Enzyme inactivated

b)  The colony we picked only contains empty vector

3.  Plasmid 5 and plasmid 6: the color of the solid LB + AMP in the petri dish turns red (because we’ve added RFP, so if the color turn red, it means that converting plasmid 5 and plasmid 6 into the E.coli succeed)  

l  PCR (to amplify the promoter of RFP ( PRFP)

- Objective: amplify the promoter of RFP

- Concepts: At 95 , the DNA helix unwinds transforme d  from double to single. When held at 72  for 10 min, all the single chains will become double. During the cycle, the structure of DNA transformed repeatedly between single and double strands.

- Materials: 

Name

Dosage μL

Mix

10

Primer: pzliE-R2-MIu

1

Primer: pzliE-Fi-Sac

1

DNA template: puc57-RFP

1

ddH2O

7

Total

20

Mix: Mg 2+ , DNA polymerase, dNTP, ddH2O 

- Methods:

9.  Mix dd water, both of the two primers, template, and the mix in a centrifuge tube ( follow the order from large quantity to small quantity )

10.  vortex and centrifuge (to avoid the occurrence of solution hanging on the wall of the tube )

11.  Place the tube inside the PCR machine

12.  Place in PCR machine

13.  Turn on the machine

14.  Click “file” on the screen

15.  Click “program

16.  Click “primmer start”

steps

temperature

time

1

95

3 min

2

95

30 s

3

55

30 s

4

72

1min

5

72

10 min

6

4

forever

PS: Repeat 2-4 30 times

Result: achieving the amplification of the promoter of plasmid 4 (PRFFP)

l  PCR (Because the DNA electrophoresis of plasmid 1 failed in the previous test, plasmid 1 should be reconstructed )  

- Objective: to amplify part of plasmid 1 (zliE-zliS )

- Concepts: At 95 , the DNA helix unwinds, changing from double to single. When held at 72  for 10 minutes, all the single chains will become double. During the cycle, the structure of DNA changes repeatedly between single and double strands.

- Materials: 

Name

Dosage μL

Mix

10

Primer: zliE-F1

1

Primer: zliS-R1

1

DNA template: zm4

1

ddH2O

7

Total

20

Mix: Mg 2+ , DNA polymerase, dNTP, dd water

- Methods: 

8.  Mix all the reagents mentioned above in a centrifuge tube ( all tubes should be placed in crushed ice, otherwise most enzyme will become inactive, such as DNA polymerase )

9.  Vortex and centrifuge (to avoid the occurrence of solution hanging on the wall of the tube )

10.  Place in PCR machine

11.  Turn on the machine

12.  Click “file” on the screen

13.  Click “program

14.  Click “primmer start”

steps

temperature

time

1

95

3 min

2

95

30 s

3

55

30 s

4

72

1min

5

72

10 min

6

4

forever

PS: Repeat 2-4 30 times

- Result: Achieving zliE-zliS amplification

l  AMP

- Objective: Cultivate E. coli with plasmid 3 while killing all E. coli that are not implanted with plasmid 3

- Concepts: The implanted E. coli is resistant to ammonia, so only the E. coli implanted with plasmamid1 and plasmid 3 can survive in the presence of AMP.

- Materials:

Name

Dosage

Stock solution (AMP)

100 mg/L

LB media

45 mL

AMP solution

60 µL

Our target concentration: 100 µg/L

100 mg/L stock solution ( AMP )

 To achieve the target concentration: 1m L  LB+1µ L  stock solution (AMP)

 Total (LB+AMP ): 5 m L /plate*20 plate=60m L  solid LB

 70 m L -->70 µ L  AMP-->80 µ L  AMP

 50 m L  centrifuge tube + 45m L  LB + 60µ L  AMP

- Methods:

1.  Spray the gloves with alcohol and check clothing on arms, hands, etc., before you put your hands in the tank (to make sure no skin is exposed to the air )

2.  Light the alcohol lamp

3.  Mix 45 m L  solid LB and 60 µL AMP in a tube

4.  Use a pipette to transfer 5 m L  of the mixture into a smaller tube

5.  Seal it and place it inside a machine to shake for a night

- Result: wait for tomorrow’s observation  

 

 

l  Gel electrophoresis 

- Objective: To check whether the DNA sample (plasmid 1 (zliE-zliS) and plasmid 4 ( promoter ) ) we have contains the correct DNA segments/fragments we need

- Concepts: The gel we constructed has a porous structure, which provides small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the correct DNA fragments we need according to the marker.

- Materials:

Name

Dosage

TAE buffer

100 mL

Agarose

1%-->1 g

YeaRed nucleic acid gel stain

1/10 thousand-->10 μL

2K marker

- Methods: (note: the containers used in this process should be marked with the words “TAE special” )

1.  Mix all reagents  mentioned above

2.  Place the solution in the microwave on medium-high heat for 2 min repeatedly until the solution boils (to ensure uniform mixing)

13.  Fix comb in the mold (thick: 50 mL; thin: 20 mL)

14.  Pour the gel

15.  Wait until it turns into a solid gel

16.  Remove the comb

17.  Pour TAE solution into the tank

18.  Use the pipette to add the four reagents made in the step “PCR” and marker to the corresponding grooves respectively.

19.  Put the lid on and power on (180V, 20 min)

20.  (After 20 min) put the gel under a gel imager to document bands

21.  Software: Genosens

22.  Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” + “collect” + “save” + name the graph (date + reagent name)

- Results: 

1.  Plasmid 1 (zliE-zliS): the line left on the gel is a little bit lower than the 2000 bp line of marker’s. target: 1680bp

2.  Plasmid 4 (promoter): the line left on the gel is a little bit higher than the 250 bp line of marker’s. target: 300bp

l  Extract plasmid 3

- Objective: Lysis of E.coli to extract plasmid 3

- Concepts: Use Buffer SP1 to degrade the RNA, then use Buffer SP2 to lyse the unwanted cytoskeleton and other wastes, then use Buffer SP3 to stop the lysis, and finally extract the Plasmid 3 in E.coli

- Materials:

Name

Dosage

Buffer S

500 µL

Water

2mL X2

Bacteria solution (E.coli with plasmid 3)

2mL X2

Buffer SP1

250 µL

Buffer SP2

250 µL

Buffer SP3

350 µL

- Methods:

1.  Add 500 µL buffer S into a tube (X2) ( Help the cells added later to better adsorb on the membrane )

2.  Centrifuge for 1 min (12 , 000 × g)

3.  Take out the tubes and pour all the solution into a garbage can

4.  Add 2mL bacteria solution (E.coli with plasmid 3 ) into a 2mL centrifuge tube, and add 2mL water to another tube for balance

5.  Centrifuge both of the two tubes for 1 min (12 , 000 × g)

6.  Take out the tubes and pour all the solution into a garbage can (some white precipitate could be observed at the bottom of the tube )

7.  Add 2 mL bacteria solution (E.coli with plasmid 3 ) into the same 2 mL tube

8.  Centrifuge both of the tubes for 1min (12 , 000 × g; also the one with water; remember: Opening towards the center of the centrifuge so that the precipitates would stay at the same spot on the tube )

9.  Take out the tubes and pour the solution in the tube with E.coli into a garbage can ( some white precipitate could be observed at the bottom of the tube )

10.  Add 250 µL buffer SP1 into the tube with E.coli, mix them by pipetting. (notion: Do not hit the bottom of the pipette and do not make bubbles; since our E.coli is a live bacteria, centrifuge is not available ) ( SP1: degrade RNA )

11.  Add 250 µL buffer SP2 into the same tube. Invert the tube back and forth 5 times to mix the solution. (SP2: lysed cells )

12.  Add 350 µL buffer SP3 into the tube. Invert the tube back and forth 5 times to mix the solution. (SP3: stop lysis; White flocs can be seen, eg: cytoskeleton )

13.  Centrifuge for 5 min (14 , 000 × g)

- Result: Separated the plasmid 3 we want from E.coli ( in the transparent solution )

l  Digestion 

- Objective: The cohesive ends on the fragments are cut out with enzymes to facilitate fusion with other fragments later. Cut out a sticky end of a fragment of gene for building up a plasmid in later process.

- Concepts: Cut out identical sticky ends to aid binding

- Materials:

 Group 1:

Name

Dosage (µL)

pUC 19 ( empty vector ) ( template for plasmid 1 )

30

Enzyme 1: Pst I-F

1

Enzyme 2: Sal I

1

rCutSmart

5

ddH2O

13

Total

50

 

Group 2:

Name

Dosage (µL)

zliES ( target gene for plasmid 1 )

30

Enzyme 1: Pst I

1

Enzyme 2: Sal I

1

rCutSmart

5

ddH2O

13

Total

50

 

Group 3:

Name

Dosage (µL)

Promoter of RFP ( target gene for plasmid 4 )

30

Enzyme 1: Sac I

1

Enzyme 2: Mlu l

1

rCutSmart

5

ddH2O

13

Total

50

 

Group 4:  

Name

Dosage (µL)

SacB ( target gene of plasmid 2 )

30

Enzyme 1: Xbal

1

Enzyme 2: Sal I

1

rCutSmart

5

ddH 2 O

13

Total

50

 

Group 5:

Name

Dosage (µL)

plasmid 3 ( the template of plasmid 4 )

30

Enzyme 1: Mlu I

1

Enzyme 2: Sac I

1

rCutSmart

5

ddH2O

13

Total

50

 

Group 6:

Name

Dosage (µL)

plasmid 1 ( the template of plasmid 2 )

30

Enzyme 1: Xbal

1

Enzyme 2: Sal I

1

rCutSmart

5

ddH2O

13

Total

50

- Method:

3.  Mix materials mentioned above in two tubes separately

c)  Group 1: pUC 19 + Pst I-F + Sal I + rCutSmart + ddH2O

d)  Group 2: zliES (target gene for plasmid 1) + Pst I + Sal I + rCutSmart + ddH2O

e)  Group 3: Promoter of RFP (target gene for plasmid 4) + Sac I + Mlu l + rCutSmart + ddH2O

f)  Group 4: SacB (target gene of plasmid 2) + Xbal + Sal I + rCutSmart + ddH2O  

g)  Group 5: plasmid 3 (the template of plasmid 4) + Mlu I + Sac I + rCutSmart + ddH2O

h)   Group 6: plasmid 1 (the template of plasmid 2) + Xbal + Sal I + rCutSmart + ddH2O  

4.  Place the tubes at a temperature of 37 ˚C for 1h

    - Result: Digestion was successfully completed and the cohesive ends were cut out.

l  DNA extraction 

- Objective: Remove proteins and other impurities that could affect results

- Concept: Elution buffer and washing buffer can help purify the genes we really want

- Materials:

Name

Dosage ( μL)

Buffer 2