Team:GDSYZX/Notebook

Notebook

7.23

Experiment Objectives

1、HMA2、 PCS1 PCR amplification
2、Detection of PCR product extraction results
3、Thermal transformation of plasmids

Experimental materials

Arabidopsis genomecDNA，Taq MIX（green），HMA2 gene primer 、PCS1 gene-specific primer 、agarose gel，TAE， goldview Stains DNAsample（Receptive cells and plasmids）、LB

Experimental procedure

①22μl MIX(green) 、1μlHMA2 gene primer、1μl Arabidopsis genomecDNA
②2μl MIX（green）、1μlPCS1 gene primer、1μl Arabidopsis genomecDNA
③PCR
④Agarose gel electrophoresis

Experimental procedure

①Add 1.5ml1μl Connected DNA sample in the centrifuge tube，Gently mix
②Place in an ice box for 30min
③Insert the floating plate，place in 42 ℃water bath for 60s
④Placed in the ice for 5min
⑤In the super-clean bench, 1ml LB culture solution (for colony growth) was added to the receptor cells and gently mix
⑥37°C Oscillatory culture for 1h（180rpm）
⑦4000rpm/s Centrifugal for three times
⑧In the super-clean bench, the top 900 milliliters of l medium was removed
⑨Blow the rest of the bacteria liquid evenly, add the solid medium (for selecting the colony) and spread the bacteria liquid evenly with the applicator
⑩37°C overnight culture

Experimental results

PCR fail

7.24

Experiment Objectives

1、Thermal transformation of plasmids
2、PCR（pcs1&hma2）

Experimental materials

Igem plasmid、 competent cell

Experimental procedure

①The competent cells were divided into eight groups and put into the centrifuge tube
②Eight groups of plasmid 5 l were added to each centrifuge tube
③Placed in the ice for 30min
④Heat-shock 42°C 60s
⑤Placed in the ice for 5min
⑥Recovery：add 600μL LB 37°C 1h
⑦coated plates： Coated with chloramphenicol medium plate and incubated at 37 C overnight

Experimental results

PCR fail

7.25

Experiment Objectives

Extration Plasmid

Experimental materials

glucose、EDTA、Rnase，NaOH，SDS、KCl、 acetic acid、Guanidine hydrochloride，Tris-HCl、Potassium acetate、absolute ethyl alcohol
Reagent formula：
solutionⅠ：50 mL Tris-HCl(pH8.0)、10 mL 1 M EDTA、the volume is constant with distilled water to 1 L
solutionⅡ：0.2 mL 10 N NaOH、1 mL 10% SDS、8.8 mLddH2O
solutionⅢ： 195.4 g Guanidine hydrochloride、37.24 g potassium acetate、61 mL acetic acid 、the volume is constant with distilled water to 500mL

Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.
Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

7.27

Experiment Objectives

1、interlab coated plates 2、PCS1 PCR(The whole genome of arabidopsis cDNA was replaced as the template) 3、pPIC9K plasmid transformation

Experimental materials

bacterium solution、competent cell 、pPIC9K

Experimental procedure

①Take 100 milligrams of l fungus solution and add it to the medium ②Apply the medium evenly with glass rods that have been burned by an alcohol lamp until the solution is dried ③37°Covernight culture

Transformation

1.Thaw 50µL competent E. coli cells on ice for 10 minutes.
2.Add:5µl DNA（pPIC9K） from a ligation reaction mix.
3.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4.Place the mixture on ice for 30 minutes. Do not mix.
5.Heat shock at exactly 42°C for exactly 60 seconds. Do not mix.
6.Place on ice for 5 minutes. Do not mix.
7.Pipette 1m l of room temperature LB media into the mixture.
8.Incubate at 37°C and 4000 rpm for 3 minutes.
9.Mix the cells thoroughly by flicking the tube and inverting.
10.Incubate overnight at 37°C with plates upside down.

Experimental results

1.PCS1 PCR succeed
2.pPIC9Ktransformation succeed

7.28

Experiment Objectives

To isolate and purify the target gene and preserve it

Experimental materials

PCR product、Binding Buffer、Wash Buffer，ddH2O

Experimental procedure

①The PCR product was electrophoresed for 45min and put into the gel imager. The gel where the target strip was cut off, and the mass was weighed and put into the centrifuge tube，take such as volume Binding Buffer.

②Place the centrifuge tube in a metal bath to melt
③Transfer the liquid to the centrifugal adsorption column，standing for 1-2min.
④12000rpm centrifuge 30s，pour the liquid out of the collecting tube
⑤Add 600μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube.
⑥Add 400μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube.
⑦Place the adsorption column into the 60 C oven lid to dry for 5min(remove the alcohol from the rinse solution) until the alcohol does not smell.
⑧Take out the adsorption column ，add 50μl ddH2O，store it.

Experimental results

Success

7.30

Experiment Objectives

1.PCS1 PCR 2.colony PCR

Experimental materials

Mix（green），cDNA，PCS1 gene primer Sterile Water 0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O

Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

Thermocycling

The PCR machine should be set to run the following steps:

7.31

Experiment Objectives

pPIC9K Extration Plasmid

Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.

Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

Experimental results

Success

8.1

Experiment Objectives

Digest（pcs1&pPIC9k）

Experimental materials

Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)
20ul Buffer 、20ul plasmid、1 ul BamHI、1 ul Not1、23 ul ddH20

Experimental procedure

Digest 37C/15 min, heat kill 80C/20 min

8.3

Experiment Objectives

1.making competent cells

Experimental materials

50ul competent cells 、1ng plasmids、LB

Experimental procedure

1、add 50ul competent cells、1ng plasmids，ice- bath for 30min 2、Put in 42 C water bath to keep warm for 1min 3、ice- bath for2 min 4、Add 1mlLB，culture at 37 degrees for 1 hour 5. Coated tablet to select

8.7

Experiment Objectives

1.colony PCR

Experimental materials

Mix（green），cDNA，PCS1 gene primer Sterile Water 0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O

Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

Thermocycling

The PCR machine should be set to run the following steps:

8.9

Experiment Objectives

1.pPIC9K Extration Plasmid

Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.
Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

Experimental results

Fail

8.12~8.14

Experiment Objectives

1.colony PCR

Experimental materials

Mix（green），cDNA，PCS1 gene primer、 Sterile Water
0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O
Note：use two kinds of primer（Species-specific primers/ Universal Primers）
Species-specific primers：（PCS1-F/PCS1-R）
Universal Primers：（AOX1-F/ AOX1-R）

Experimental procedure

Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
1.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

Thermocycling

The PCR machine should be set to run the following steps:

8.15

Experiment Objectives

1.Pcs1 PCR

Experimental materials

Mix（green），pcs1， coPCS1F,coPCS1R

Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

Thermocycling

3.Agarose gel electrophoresis
4.gum concentration:15ng
5.Separation and purification

materials

PCR product、Binding Buffer、Wash Buffer，ddH2O

procedure

⑨The PCR product was electrophoresed for 45min and put into the gel imager. The gel where the target strip was cut off, and the mass was weighed and put into the centrifuge tube，take such as volume Binding Buffer
⑩Place the centrifuge tube in a metal bath to melt
⑪Transfer the liquid to the centrifugal adsorption column，standing for 1-2min
⑫12000rpm centrifuge 30s，pour the liquid out of the collecting tube
⑬Add 600μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube
⑭Add 400μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube
⑮Place the adsorption column into the 60 C oven lid to dry for 5min(remove the alcohol from the rinse solution) until the alcohol does not smell
⑯Take out the adsorption column ，add 50μl ddH2O，store it

8.16

Experiment Objectives

Digest（pcs1&pPIC9k）

Experimental materials

Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns) 20ul Buffer 、20ul plasmid、1 ul BamHI、1 ul Not1、23 ul ddH20

Experimental procedure

Digest 37C/15 min, heat kill 80C/20 min

Experiment Objectives

ligation（pcs1&pPIC9k）

Experimental materials

pPIC9k、pcs1、 T4 DNA ligase buffer、T4 DNA ligase

Experimental procedure

1.Add 9.5ul pPIC9k、7.5 ul pcs1、2 ul T4 DNA ligase buffer、0.8 ul T4 DNA ligase
2.Ligate 16~25℃/4h, heat kill 80C/20 min
3.Transform with 1-2 ul of product

8.17

Experiment Objectives

Transformed recombinant vector

Experimental materials

NEB competent cells

Experimental procedure

1.Add 50ul competent cells，10ulNEB,，ice-bath for 30min，42℃ for 1min
2.Add 200ulSOC Medium，37℃ recover for1h

8.18

Experiment Objectives

ppic9k（colony pcr）

Experimental materials

Mix（green），cDNA，PCS1 gene primer、 Sterile Water
0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O
Note：use two kinds of primer（Species-specific primers/ Universal Primers）
Species-specific primers：（PCS1-F/PCS1-R）
Universal Primers：（AOX1-F/ AOX1-R）

Experimental procedure

Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.

1.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

Thermocycling

The PCR machine should be set to run the following steps:

8.19

Experiment Objectives

Transformation

Experimental materials

LB broth、Ice、Selection plates

Experimental procedure

1.Thaw 50µL competent E. coli cells on ice for 10 minutes.
2.Add:1µl DNA（pcs1&pPIC9K） from a ligation reaction mix.
3.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4.Place the mixture on ice for 30 minutes. Do not mix.
5.Heat shock at exactly 42°C for exactly 60 seconds. Do not mix.
6.Place on ice for 5 minutes. Do not mix.
7.Pipette 1m l of room temperature LB media into the mixture.
8.Incubate at 37°C and 4000 rpm for 3 minutes.
9.Mix the cells thoroughly by flicking the tube and inverting.
Incubate overnight at 37°C with plates upside down.

Experiment Objectives

Extration Plasmid

Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.
Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

8.20

Experiment Objectives

ppic9k-copcs1（colony pcr）

Experimental materials

Mix（green），cDNA，PCS1 gene primer、 Sterile Water 0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O Note：use two kinds of primer（Species-specific primers/ Universal Primers） Species-specific primers：（PCS1-F/PCS1-R） Universal Primers：（AOX1-F/ AOX1-R）

Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells. 2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

Thermocycling

The PCR machine should be set to run the following steps:

8.21

Experiment Objectives

1.Pcs1 pcr 2.pPIC9K-copcs1 (colony pcr) 3.competent yeast

Experimental materials (making competent yeast)

PEG(50%)、Licl、linear TtDNA、DNA,FishSperm

Experimental procedure

1.Add 1ml connection product into 1.5ml centrifugal tube
2.2000rpm centrifuge 5min
3.Throw away the supernatant
4.Add 1ml ddH20
5.2000rpm centrifuge 5min
6.Throw away the supernatant
7.Add 300μl ddH20
8.2000rpm centrifuge 5min
9.500μl 0.1M Licl ,4000rpm centrifuge 3min
10.Throw away the supernatant
11.Add 200μl PEG(50%)、30μl 1M Licl 、20μl DNA,FishSperm、50μl linear TtDNA
12.Standing 30℃ 30min，42℃ 25min
13.7000rpm centrifuge 3min
14.Throw away the supernatant
15.Add 500μl ddH20

Experimental materials(Transform into the yeast)

Competent yeast 、PEG、TE 、MD

Experimental procedure

1.Add 50μl competent yeast、2μl plasmids(pPIC9K-copcs1)MIX
2.Add 500μl (PEG/LiAc,DMSO）MIX
3.Ice-bath at 30℃ for 1h，mix every 15min
4.Add 1ml YPD，cultivate in shaker at 30℃ for 1h
5.3500rpm centrifuge 5min，throw away the supernatant
6.Add 150μl TE，coated plates in MD
7.30℃ cultivate

8.22

Experiment Objectives

Yeast resistance in cdcl2 test（3 methods）

Experimental materials

Cdcl2、yeast、YPD

Experimental procedure

1.Add 100μl 0.5mM Cdcl2 in YPD，use ss-spreader to spread evenly×3
2.Add 100μl 0.6mM Cdcl2 in YPD，use ss-spreader to spread evenly×3
3.Add 100μl 0.7mM Cdcl2 in YPD，use ss-spreader to spread evenly×3
4.Add 100μl 0.8mM Cdcl2 in YPD，use ss-spreader to spread evenly×3
5.Add 100μl 0.9mM Cdcl2 in YPD，use ss-spreader to spread evenly×3
6.Add 100μl 1mM Cdcl2 in YPD，use ss-spreader to spread evenly×3

Experimental procedure

1.Mix Cdcl2 、yeast first(0.5mM Cdcl2、0.6mM Cdcl2、0.7mM Cdcl2、0.8mM Cdcl2 、0.9mM Cdcl2 、1mM Cdcl2)
2.Use ss-spreader to spread evenly

Experimental procedure

1.Add 100μl yeast（with different concentration） in YPD, cultivate for 8h
2.Dropwise added 2μl Cdcl2 on the yeast

Note

Different concentration：0.5mM Cdcl2、0.6mM Cdcl2、0.7mM Cdcl2、0.8mM Cdcl2 、0.9mM Cdcl2 、1mM Cdcl2

8.23

Experiment Objectives

Making yeast growth curve

Experimental materials

Yeast（mother liquid）、YPD（Solid）

Experimental procedure

1.Dilute the yeast（mother liquid）with water to different concentration.
Concentration: 10-2 、10-3、10-4 、10-5、10-6
2.Coat the yeast（with different concentration）on YPD medium，cultivate it.
3. Count the number of the colony
4. Make the yeast growth curve

8.24~8.25

Experiment Objectives

1.PAGE（proteins electrophoresis, ）

Experimental materials

30%Acr、10%SDS、1.5mol/L pH8.8 Tris-Hcl Buffer、1.0mol/L pH6.8 Tris-Hcl Buffer、0.05mol/L pH8.0 Tris-Hcl Buffer、10%AP、TEMED、sample solution、staining solution、destaining solution、 coagulant

Experimental procedure

1.Cleaning the glass plate
2.Filling the glass plate with buffer and coagulant
3.Loading the sample
4. Electrophoresis
5.Staining&decolorize