Team:Gifu/Protocol

　

PROTOCOL

Medium

LB medium(100ml)
Trypton10g
Yeast Extract0.5g
NaCl1.0g
H2O100ml
agar (in case of agar medium)2.0g

SOC medium(100mL)

Tryptone2.0g
Yeast Extract0.5g
1M NaCl1.0mL
1M KCl0.25mL
H2O(up to 100mL)

YPD medium(100mL)

Yeast Extract1.0g
Glucose2.0g
Peptone2.0g
H2O100mL

Minimal medium for E.coli(DM medium)

K2HPO40.7g
KH2PO40.3g
MgSO4・7H2O0.01g
Glucose0.2g
H2O100mL

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


Minimal medium for S.cerevisiae and S.pombe

Yeast extract nitrogen base(w/o ammonia and amino acids)1.7g
Glucose1.0g
H2O100mL

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


Medium for Bacillus subtilis

Hipolypeptone1g
Yeast Extract0.2g
MgSO4·7H2O0.1g
H2O100mL
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.