Difference between revisions of "Team:Gifu/Protocol"

• 
  Project
  • Abstract
  • Back ground
  • Purine metabolism
  • Experiments
  • Results
  • Conclusion
  • Modeling
  • Futurework
  • Reference
• 
  Lab Note
  • Protocol
  • Calendar
  • InterLab
  • Proof
• 
  Parts
  • Basic Part
  • Composite Part
  • Part Collection
• Human Practices
  • Human Practices
• 
  About Us
  • Team
  • Collaborations
  • Safety
  • Attributions

　

PROTOCOL

---

---

Medium

SOC medium(100mL)

Tryptone	2.0g
Yeast Extract	0.5g
1M NaCl	1.0mL
1M KCl	0.25mL
H2O	(up to 100mL)

YPD medium(100mL)

Yeast Extract	1.0g
Glucose	2.0g
Peptone	2.0g
H2O	100mL

Minimal medium for E.coli(DM medium)

K2HPO4	0.7g
KH2PO4	0.3g
MgSO4・7H2O	0.01g
Glucose	0.2g
H2O	100mL

(Add 2mM Uric acid or allantoin or ammonium sulfate as a sole source.)

Minimal medium for S.cerevisiae and S.pombe

Yeast extract nitrogen base(w/o ammonia and amino acids)	1.7g
Glucose	1.0g
H2O	100mL

(Add 2mM Uric acid or allantoin or ammonium sulfate as a sole nitrogen source.)

Medium for Bacillus subtilis

Hipolypeptone	1g
Yeast Extract	0.2g
MgSO4·7H2O	0.1g
H2O	100mL
Agar (if needed)	1.5g

Restriction digests

Materials

Ice and a bucket/a container
DNA to be digested
CutSmartTM buffer
Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
Incubater

Procedure (an example)

1.Add 43ul of DNA to be digested into a 1.5ml microcentrifuge tube.
2.Add 5.0ul of 10x CutSmartTM buffer.
3.Add 1.0ul of EcoRI.
4.Add 1.0ul of SpeI.
5.There should be a total volume of 50ul. Mix well and spin down briefly.
6.Incubate the restriction digest at 38℃ for 30min.
7.Run a portion of the digest on a gel (6ul) and check that part length is accurate.

Ligation

1. Add digested fragment A.
2. Add digested fragment B.
3. Add ligation mixture.
4. Ligation 16℃ for 30 min.
Note: Make the amount of fragment A and B equimolar. The volume of ligation mixture is equivalent to the total volume of the mixed solution of fragment A and B. We used a constant temperature water bath.

Transformation

1.Dispense 25ul of competent cells to each 1.5ml tubes.
2.Add 1ul of DNA to each tube.
3.Close tubes and incubate the cells for 30 min on ice.
4.Heat shock the cells at 42℃ for 60 seconds.
5.Incubate the cells on ice for 2 minutes.
6.Add 225ul of SOC medium to each tube.
7.Incubate cells at 37℃ for 30 minutes.
8.Spread 50μL of cells onto each plate containing appropriate antibiotics.
9.Culture the cells.

DNA densitometry(NanoDrop)

1.Activate the device.
2.Choose “Nucleic Acid” at main menu.
3.Wash the upper and lower optical surfaces of the microspectrophotometer sample retention system with distilled water. Wipe off both optical surfaces with a Kimwipe gently.
4.Choose a sample type. There are DNA-50(double-strand), DNA-33(single-strand), RNA.
5.Pour 1μL distilled water to test a blank and clean both optical surfaces with a Kimwipe
6.Pour 1μL of the sample to measure the concentration.

Colony PCR

Materials

Prepare reaction mixture(45μL)

Nuclease free water	27.5 µL
10×PCR buffer	5μL
2mM dNTP	5μL
5 pmol/µL Fw primer	2.5μL
5 pmol/µL Rv primer	2.5μL
0.5U/μL Taq polymerase	2.5μL

Procedure

1. Pick a colony on a plate and suspend it in 100μL of LB medium(liquid)
2. Add 5μL of diluted cell culture to reaction mixture.(Total volume is 50μL)
3. Do PCR amplification.
4. Measure the length of the DNA by Electrophresis.

Electrophoresis

Materials

Prepare 100mL of agarose solution

Agarose	2g
50×TAE buffer	2mL
Deionized water	98mL

Procedure

1. Heat agarose solution in a microwave oven to dissolve agarose completely.
2. Cool the solution to an appropriate temperature.
3. Pour the solution into a mold with a comb.
4. Remove bubbles in the solution.
5. After the gel harden, remove the comb carefully.
6. Transfer the mold into a phoresis tank.
7. Immerse the mold in 1×TAE buffer.
8. Mix 5 µL of DNA solution and 1 µL of 6× loading buffer.
9. Pour the mixture into the well.
10. Electrophorese at 100V.
11. Stop the electrophoresis when the band of dye move up to 3/4 of the gel.
12. Pick out the gel and then stain it with ethidium bromide (0.5 µg/mL) for 20 minutes.
13. Observe DNA bands with UV transilluminator.

Miniprep

Materials

Prepare following solutions

SolⅠ: 50mM glucose/25mM Tris-HCl/10mM EDTA
SolⅡ: 0.2N NaOH/1%SDS
SolⅢ: 3M acetic acid/5M potassium

Procedure

1. Dispense 1000μL of culture to a 1.5ml tube.
2. Centrifuge(14,000rpm,room temperature,1minutes) and remove the top layer by decantation.
3. Add 50μL of SolⅠand mix it by vortex.
4. Add 100μL of SolⅡ and mix it very slowly and put it gently on ice for 1minute.
5. Add 75μL of SolⅢ and mix it slowly and put it gently on ice for 5minutes.
6. Centrifuge(14,000rpm,4℃,5minutes) and move the top layer to a new tube.
7. Add 225μL of chloroform/phenol(1:1) and mix it .
8. Centrifuge(14,000rpm,room temperature,10minutes) and move the top layer to a new tube.
9. Add 225μL of chloroform and mix it.
10. Centrifuge(14,000rpm,room temperature,5minutes) and move the top layer to a new tube.
11. Add 225μL of propan-2-ol and mix it and put it gently for 5 minutes.
12. Centrifuge(14,000rpm,room temperature,10minutes) and remove the top layer.
13. Add 500μL of 70% ethanol and turn upside-down to clean the inside of the tube.
14. Centrifuge(14,000rpm,room temperature,5minutes) and remove the top layer completely and dry it by vacuum drying.
15. Melt the precipitation by adding 20μL of 20μg/mL RNase A/TE buffer.
16. Incubate it for 30minutes in 37℃.

Gel Extraction with FastGene™ Gel/PCR Extraction Kit

Sample preparation

1.Cut down a DNA fragment from an agarose gel. Remove surplus agarose to make the gel fragment as small as possible.
Note: Recommended concentration of agarose is under 2.5%.
2.Transfer the gel fragment (up to 300 mg of gel) to a centrifugal tube.
3.Add 500 µL of GP1 to a sample and voltex the tube.
4.Incubate the sample at 55℃for 10~15 minutes (until the gel fragment completely dissolves). Invert the tube every 2~3 minutes while incubating.

Sample loading

1.Insert FastGene™GP column into a collection tube.
2.Dispense (up to 800 µL of) sample solution prepared previously into FastGene™GP columns　and then centrifuge it(13,000 rpm 30 seconds).
3.Throw filtrate away and then return the column to the collection tube.
4.Caution: If the volume of the sample solution is over 800 µL, repeat the step of DNA bonding.

Membrane washing

1.Add 600 µL of GP2 to the column and then centrifuge it(13,000 rpm 30 seconds).
2.Throw filtrate away and then return the column to the collection tube.
3.Caution: If you use TAE gel, proceed to the next step. If you use TBE gel, repeat this step to remove boric acid completely.

Membrane drying

1.Centrifuge a column matrix(13,000rpm 2 minutes) to desiccate it.

DNA Elution

1.Insert a FastGene™GP column into a new centrifugal tube.
2.Add 20~50 µL of elution buffer GP3 to the center of column matrix.
3.Keep the same state for 2 minutes at room temperature to ensure GP3 is absorbed by the column matrix,.
4.Centrifuge it(13,000 rpm 2 minutes) and then elute refined DNA.

PCR Purification with FastGene™ Gel/PCR Extraction Kit

Sample preparation

1.Mix GP1 and PCR products in the ratio of 5 to 1 in a centrifuge tube(e.g. 200 µL of GP1 per 40 µL of PCR products).
Caution: If the volume of the sample is under 40 µL, add GP1 or water for PCR products to adjust the volume of the sample to 40 µL.

Sample loading

1.Insert FastGene™GP column into a collection tube.
2.Dispense (up to 800 µL of) sample solution prepared previously into FastGene™GP columns　and then centrifuge it(13,000 rpm 30 seconds).
3.Throw filtrate away and then return the column to the collection tube.
4.Caution: If the volume of the sample solution is over 800 µL, repeat the step of DNA bonding.

Membrane washing

1.Add 600 µL of GP2 to the column and then centrifuge it(13,000 rpm 30 seconds).
2.Throw filtrate away and then return the column to the collection tube.
3.Caution: If you use TAE gel, proceed to the next step. If you use TBE gel, repeat this step to remove boric acid completely.

Membrane drying

1.Centrifuge a column matrix(13,000rpm 2 minutes) to desiccate it.

DNA Elution

1.Insert a FastGene™GP column into a new centrifugal tube.
2.Add 20~50 µL of elution buffer GP3 to the center of column matrix.
3.Keep the same state for 2 minutes at room temperature to ensure GP3 is absorbed by the column matrix,.
4.Centrifuge it(13,000 rpm 2 minutes) and then elute refined DNA.

HisLink Spin Protein Purification

Materials to Be Supplied by the User

・Nuclease-Free Water
・rotor
・wide-bore pipette tips
・5M NaCl solution
・tabletop centrifuge
・1.5ml microcentrifuge tubes

Preparation of FastBreak™ Reagent/DNase I solution

1.Add 80μL of Nuclease-Free Water to the vial of DNaseⅠ.
2.Mix completely to dissolve the powder.
3.Remove the DNase solution from the vial and add it to 1mL of Nuclease-Free Water.
Mix well.
4.Mix 5.8μL of the FastBreak™ Reagent and 64.2μL of the DNase I solution.

Procedure of centrifugation

1.Pipet 700μl of bacterial culture into a 1.5ml microcentrifuge tube. Add 70μl of the FastBreak™ Reagent/DNase I solution.
2.Resuspend the resin and allow it to settle. Once the resin has settled, use a wide-bore pipette tip to transfer 75μl of the HisLink™ Resin from the settled resin bed to the 1.5ml microcentrifuge tube. To successfully transfer resin, place the wide-bore pipette tip deep into the resin and pipet slowly to assure that a consistent amount of resin is drawn into the pipette. Allow the resin to resettle between each pipetting.
Note: We recommend optimizing the amount of HisLink™ Resin used for low- (<1mg/ml) or high- (>1mg/sample) expressing proteins. For low-expressing proteins, less resin should be used; similarly, for high-expressing proteins, more resin per sample can be used.
3.Incubate the sample and resin for 30 minutes, mixing frequently on a rotating platform or shaker to optimize binding.
4.Place a Spin Column onto a Collection Tube (or a new 1.5ml microcentrifuge tube). Use a wide-bore pipette tip to transfer the lysate and resin from the original 1.5ml microcentrifuge tube in Step 3 to the spin column. If resin remains in the 1.5ml microcentrifuge tube, add HisLink™ Binding/Wash Buffer to the tube, then transfer the buffer and remaining resin to the spin column.
5.Centrifuge the spin column with the collection tube for 5 seconds or until the liquid clears the spin column.
6.To save the flowthrough, remove the spin column from the collection tube and transfer the flowthrough from the collection tube to a new 1.5ml microcentrifuge tube. Otherwise, discard the flowthrough.
7.Place the spin column back onto the collection tube. Add 500μl of HisLink™ Binding/Wash Buffer to the spin column, then cap the spin column. Centrifuge for 5 seconds or until the Binding/Wash Buffer clears the spin column. Discard the flowthrough. Repeat for a total of two washes.
8.Take the spin column off the collection tube and wipe the base of the spin column with a clean absorbent paper towel to remove any excess HisLink™ Binding/Wash Buffer.
9.Place the spin column onto a new 1.5ml microcentrifuge tube. Add 200μl of HisLink™ Elution Buffer. Cap the spin column and tap or flick it several times to resuspend the resin. Wait 3 minutes.
10.Centrifuge the spin column and microcentrifuge tube at 14,000rpm for 1 minute to collect the eluted protein.

SDS-PAGE

Protein extraction and Sample preparation

1.Add 50 µL of cell suspension that was cultured overnight to LB medium, incubate at 37℃ for two hours.
2.Measure the turbidity of E. coli culture at a wavelength of 660nm. Dilute the cell suspension with LB liquid and make the optical density(660nm) equivalent to 0.5. Add IPTG to the cell suspension.
3.Cultivate it for 3 hours and then store it at low temperature.
4.Dispense 1 mL of the suspension into a 1.5ml tube. Centrifuge it(13,000 rpm 4°C 15 minutes) and then remove the supernatant .Repeat this step twice.
5.Add 300 µL of PBS to the deposition and then mix it.
6.Sonicate the cell suspension for 15 sec and cool it for 60 sec on ice. Repeat this step 4times.
7.Centrifuge(13,000rpm, 4°C, 20minutes)and transfer the supernatant into another tube.
8.Add 100 μL of PBS to the deposition. Vortex the tube.
9.Dispense 8 µL of the supernatant(=cell extract) into a new tube and 8 µL of the precipitation suspension into another tube.
10.Add 2 µL of 5×loading dye to the tubes and then denature it at 90℃.
11.Centrifuge and prepare the sample.
12.Apply 10µL of the sample to SDS gel.

Making a SDS-PAGE gel and Electrophpresis

1 Mix and shake reagents quickly to prepare the separation gel.
2 Construct a plate for electrophoresis.
3 Pour separation gel into a gap of the plate (until about 2 cm below a comb).
Note: Wipe a plate for electrophoresis with 70% ethanol.
Hold a plate for electrophoresis not to spill the gel.
4 Pour a proper quantity of Milli Q water into a gap of the plate and then incubate for an hour at room tempareture.
5 Mix and shake quickly reagents for stacking gel (except APS and TEMED).
6 Slant the gel plate and absorb multistoried Milli Q water.
7 Add APS and TEMED to the mixture. (step5)
8 Fill the gap of the plate with stacking gel and then insert the comb into the gap of the plate.
Note: Be careful not to generate bubbles in gel.
9 Take out the plate and gel together after stacking gel coagulates.
10 Put the plate and gel into a migration tank with the plate toward outside.
11 Pour 300 µL of electrophoresis buffer into a phoresis tank. Immerse the gel completely.
12 Apply 10 µL of the sample and 5 µL of a marker.
13 Electrophorese at 40 mA in the stacking gel until a pigment comes at the separation gel.
After that, electrophorese at 60mA in the separation gel.
14 Stop electrophoresis and then collect the gel carefylly.
Note: Use tweezers.
15 Remove the buffer for electrophoresis and dye the separation gel with CBB.
16 Wash the gel plate and the electrophoretic tank with neutral detergent and rinse it steadily.

Staining with CBB

1 Put the gel into fixing solution.
2 Leave the gel on the shaker until a band is dyed yellow.
3 Remove the fixing solution and then put the gel into CBB dyeing liquid.
4 Wrap it and then heat it until it is almost boiling with a microwave oven.
5 Remove the wrap carefully and then let vapor out slowly.
6 Remove the CBB dyeing liquid and pour deionized water into a container carrying the gel. Put Kim wipe into the container.
7 Infiltrate deionized water into the gel for several tens of minutes. Transfer waste liquid into a tank.
Take a picture under UV light and then dry the gel and store it.