PROTOCOL
       

Protocols

Transformation

Material
Z-Competent E. coli Transformation Kit and Buffer set (funakoshi)

Method
1. Put the competent cell on ice and leave it until disolution.
2. Add DNA 1µL to competent cell 25µL on 2ml tube.
3. Place on ice for 20min.
4. Heatshock at 42℃ for 45sec.
5. Place the tube on ice for 3 minutes.
6. Add 100µL of LB medium and place tube on 37°C for 20min.
7. Plate 100µL of the medium and spread well.
8. Incubate the plate on 37℃.

Electrophoresis

Method
1. Make a gel for electrophoresis
2．Add Agarose to 30ml or 15ml of 1xTAE buffer in Erlenmeyer flask per 1 gel plate. The concentration of gel is 1% for over 1kbp of DNA and use 2% to under 1kbp.
3．Wrap the top of Erlenmeyer Flask not to completely seal up.
4．Heat it by microwaves until the solution gets transparent.
5．Pour it into a maker and set a comb before it get hard.
6．After the gel get hard , remove the comb carefully not to break well.
7．Place maker into tank for electrophoresis and fill 1xTAE up to over the level of gel .
8. Add 2µl of 6xLoading buffer into 10µl of DNA solution and vortex it.
9. Apply 12µl of sample and 6µl of ladder solution in each well.
10. Electrophoresis at 100V for 20-30 minutes.
11. The marker band reaches 80% of the gel, stop electrophoresis and salvage the gel.
12. Put it to the ethidium bromide solution for 15 min for staining.
13. Check the band under blue light.

Colony PCR

Method
1.
2. Dispense it to each PCR tube for 10µl on ice.
3. Pick up single colony on plate and dip to the tube.
4. Set to Thermal cycler.
5．Check the length of DNA by Electrophoresis.

Miniprep

Material
Wizard® Plus SV Minipreps DNA Purification System (promega)
Method
1. Pour 3ml of LB liquid to test tube.
2. Pick up a single colony and put it into the tube.
3. Incubate it at 37℃ for 8 hours with the tube shaking.
4. Transfer 1.5ml of culture fluid to 1.5ml tube.
5. Centrifuge at 15000rpm for 1minutes.
6. Throw away the supernatant and add the other 1.0ml.
7. The same as 5 and 6 and be only precipitation.
8. Add 250µl of Cell-Resuspension Sol and Vortex it until precipitation dissolved.
9. Add 250µl of Cell-Lysis Sol and invert it not to make bubble.
10. Add 10µl of Alkaline Protease and invert it not to make bubble
11. Leave it for a few minutes at room temperature.
12. Add 350ml of Neutralization Sol and invert it not to make bubble.
13. Centrifuge at 15000rpm 10min.
14. Set a collection tube on column and apply sup to it.
15. Centrifuge at15000rpm for 1min.
16. Add 750µl of Column Wash Sol and Centrifuge for 1min at 15000rpm.
17. Add 250µl of Column Wash Sol and Centrifuge for 1min at 15000rpm.
18. Centrifuge for 2min at 15000rpm to dry the column.
19. Transfer the column to another 1.5ml tube.
20. Add 20ml of nuclease-free water and leave it for 1min at room temperature.
21. Centrifuge for 1min at 15000rpm.
22. Measure concentration of flow though liquid by using Nano Drop.

Digestion

Material
The restriction enzymes (EcoRⅠ, XbaⅠ, SpeⅠ and PstⅠ) are purchased or provided as support for iGEM Japan from Promega.
Method
1. Mix DNA solution and reagent as follows.(unit µl)
2. Incubate for 3~6 hours at 37℃.
3. Add 4µl of 6x Loading Buffer and Electrophoresis and Gel Extraction.

Gel extraction

Method
1. After electrophoresis, cut the gel including DNA and put it into 1.5ml tube.
2. Add (900 – 150* the number of fragments of gel)µl of Membrane Bind Sol and stand at 60℃(Vortex per 2min)
3. Apply to column-set collection tube and Centrifuge for 1min at 12000rpm.
4. Add 750µl of Membrane Wash Sol to the column.
5. Centrifuge for 1min and remove flow through.
6. Add 500µl of Membrane Wash Sol to the column.
7. The same way as 5.
8. Centrifuge for 2min to dry the column.
9. The same way as method of Miniprep 19~22.

Ligation

Method
1. Calculate the mass ratio of Insert DNA to Vector DNA to equal
2. Mix the DNA solution and reagent as follows.
3. Stand it for 15min at room temperature.

qRT-PCR

Method
1. Pick up a colony and inoculate in LB broth containing 100µg/mL ampicillin.
2. Incubate at 37 degree over night.
3. Dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin.
4. Grow to OD600=0.6 at 37 degree.
5. Centrifuge 1mL culture at 15,000rpm for 1min.
6. Remove the supernatant.
7. Resuspend with TE buffer containing lysozyme(0.4mg/mL).
8. Incubate at room temperature for 5min.
9. Isolate total RNA from the lysate with RNeasy® Mini.
10. Synthesize cDNA from 1µg total RNA with PrimeScript® 1st strand cDNA Synthesis Kit.
11. Quantify the cDNA of the target gene or 16S rRNA with Power SYBR® Green PCR Master Mix.