Team:Gifu/Protocol
PROTOCOL
Medium
LB medium(100 mL)
| Tryptone | 10g |
| Yeast Extract | 0.5g |
| NaCl | 1.0g |
| H2O | 100 ml |
| agar (in case of agar medium) | 2.0g |
SOC medium(100 mL)
| Tryptone | 2.0g |
| Yeast Extract | 0.5g |
| 1M NaCl | 1.0 mL |
| 1M KCl | 0.25 mL |
| H2O | (up to 100 mL) |
YPD medium(100 mL)
| Yeast Extract | 1.0g |
| Glucose | 2.0g |
| Peptone | 2.0g |
| H2O | 100 mL |
Minimal medium for E. coli (DM medium)
| K2HPO4 | 0.7g |
| KH2PO4 | 0.3g |
| MgSO4・7H2O | 0.01g |
| Glucose | 0.2g |
| H2O | 100 mL |
(Add 2 mM 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 2 mM 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 | 100 mL |
| 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 43 ul of DNA to be digested into a 1.5 ml microcentrifuge tube.
2.Add 5.0 ul of 10x CutSmartTM buffer.
3.Add 1.0 ul of EcoRI.
4.Add 1.0 ul of SpeI.
5.There should be a total volume of 50 ul. Mix well and spin down briefly.
6.Incubate the restriction digest at 38℃ for 30 min.
7.Run a portion of the digest on a gel (6 ul) 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 25 ul of competent cells to each 1.5 ml tubes.
2.Add 1 ul 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 225 ul of SOC medium to each tube.
7.Incubate cells at 37℃ for 30 minutes.
8.Spread 50 μL 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 1 μL distilled water to test a blank and clean both optical surfaces with a Kimwipe
6.Pour 1 μL of the sample to measure the concentration.
Colony PCR
Materials
Prepare reaction mixture(45 μL)
| Nuclease free water | 27.5 µL |
| 10×PCR buffer | 5 μL |
| 2 mM dNTP | 5 μL |
| 5 pmol/µL Fw primer | 2.5 μL |
| 5 pmol/µL Rv primer | 2.5 μL |
| 0.5 U/μL Taq polymerase | 2.5 μL |
Procedure
1. Pick a colony on a plate and suspend it in 100 μL of LB medium(liquid)
2. Add 5 μL of diluted cell culture to reaction mixture.(Total volume is 50 μL)
3. Do PCR amplification.
4. Measure the length of the DNA by Electrophresis.
Electrophoresis
Materials
Prepare 100 mL of agarose solution
| Agarose | 2g |
| 50×TAE buffer | 2 mL |
| Deionized water | 98 mL |
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 100 V.
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: 50 mM glucose/25 mM Tris-HCl/10 mM EDTA
SolⅡ: 0.2N NaOH/1%SDS
SolⅢ: 3M acetic acid/5M potassium
Procedure
1. Dispense 1000 μL of culture to a 1.5 ml tube.
2. Centrifuge(14,000rpm, room temperature,1 minutes) and remove the top layer by decantation.
3. Add 50 μL of SolⅠand mix it by vortex.
4. Add 100 μL of SolⅡ and mix it very slowly and put it gently on ice for 1 minute.
5. Add 75 μL of SolⅢ and mix it slowly and put it gently on ice for 5 minutes.
6. Centrifuge(14,000rpm, 4℃, 5 minutes) and move the top layer to a new tube.
7. Add 225 μL of chloroform/phenol(1:1) and mix it .
8. Centrifuge(14,000rpm, room temperature, 10 minutes) and move the top layer to a new tube.
9. Add 225 μL of chloroform and mix it.
10. Centrifuge(14,000rpm, room temperature, 5 minutes) and move the top layer to a new tube.
11. Add 225 μL of propan-2-ol and mix it and put it gently for 5 minutes.
12. Centrifuge(14,000rpm, room temperature, 10 minutes) and remove the top layer.
13. Add 500 μL of 70% ethanol and turn upside-down to clean the inside of the tube.
14. Centrifuge(14,000rpm, room temperature, 5 minutes) and remove the top layer completely and dry it by vacuum drying.
15. Melt the precipitation by adding 20 μL of 20 μg/mL RNase A/TE buffer.
16. Incubate it for 30 minutes 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(13,000rpm, 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(13,000rpm, 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(13,000rpm, 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(13,000rpm, 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(13,000rpm, 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(13,000rpm, 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
・1.5 ml microcentrifuge tubes
Preparation of FastBreak™ Reagent/DNase I solution
1.Add 80 μL 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 1 mL of Nuclease-Free Water.
Mix well.
4.Mix 5.8 μL of the FastBreak™ Reagent and 64.2 μL of the DNase I solution.
Procedure of centrifugation
1.Pipet 700 μl of bacterial culture into a 1.5 ml microcentrifuge tube. Add 70 μl 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 75 μl of the HisLink™ Resin from the settled resin bed to the 1.5 ml 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- (<1 mg/ml) or high- (>1 mg/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 1.5 ml microcentrifuge tube). Use a wide-bore pipette tip to transfer the lysate and resin from the original 1.5 ml microcentrifuge tube in Step 3 to the spin column. If resin remains in the 1.5 ml 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 1.5 ml microcentrifuge tube. Otherwise, discard the flowthrough.
7.Place the spin column back onto the collection tube. Add 500 μl 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 1.5 ml microcentrifuge tube. Add 200 μl 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 660 nm. Dilute the cell suspension with LB liquid and make the optical density(660 nm) 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 1.5 ml tube. Centrifuge it(13,000rpm, 4°C, 15 minutes) and then remove the supernatant .Repeat this step twice.
5.Add 300 µL of PBS to the precipitation 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, 20 minutes)and transfer the supernatant into another tube.
8.Add 100 μL of PBS to the precipitation. 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 10 µL 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 60 mA 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.
