Team:Warsaw/protocols

introduction
Here is the place for the all protocols used by the Warsaw Team during the iGEM 2009. Feel free to add yours.

Chemotransformation

 * thaw bacteria on the ice - 10 minuts
 * Add desired volume of DNA to the 100-μl-culture in eppendorf tube
 * incubation on the ice - 20 minutes
 * heat shock - 1 minut, 42&deg;C
 * incubation on the ice - 2 minuts
 * add 900 ul of SOB medium to the bacteria
 * incubation in 37&deg;C - 1 h
 * Plating on appropriate medium

Plasmid DNA isolation
We use "Plasmid Mini" plasmid DNA isolation kit from A&A Biotechnology and follow the protocol of the producer.

DNA isolation from agarose gel
We use "Gel-Out" DNA isolation kit from A&A Biotechnology and follow the protocol of the producer. We also use similar kit from EURx.

DNA purification after enzymatic reaction
We use "Clean-Up" DNA purification kit from A&A Biotechnology and follow the protocol of the producer.

Genomic DNA isolation
We use "Genomic-Mini" universal genomic DNA isolation kit from A&A Biotechnology and follow the protocol of producer.

DNA digest
We use restriction enzymes and buffers provided by Fermentas. Overall volume of digest mix is either 20 μl, either 50 μl in case of digesting for ligation. We usually use 1 μl of restriction enzyme and the buffer in 10x dilution (as they initially are 10x concentrated). The rest of mix is plasmid DNA.

Preparation of glycerol stocks
1) Centrifuge the liquid bacterial culture at 6 000 rpm for 2 minutes.

2)Suspend in glicerol-LB solution (20% glycerol - 80%LB volume solution).

3) Mix by pipetting or gently vortex.

4) Write the name of the strain or some useful identifier on the top of the eppendorfs.

5) Pour solution into the eppendorfs max. 1 ml per eppendorf, because the volume will expand.

6) Incubate on ice for 15 min.

7) Store in -80C freezer

Alkaline lysis
Perform on ice, centrifuge in 4°C . Solution: 1,2,3 and 70% ethanol solution are prepared in advance. Solution 1 and 3 are stored in the refrigerator. Solution 2 should be stored at room temperature, no longer than 4-6 days. Solution should be mixed by inverting the tubes before use. The volumes of the solutions in this protocol are for the alkaline lysis of the pellet from 3ul overnight culture of E.Coli. For the pellet from 5 μl use greater volumes.

Solutions
Solution 1 -

Solution 2 - NaOH 0.2M, SDS 1%

Solution 3 -

70% ethanol solution

Procedure
1.Centrifuge the cultures at 6000 rpm for 5min.

2. Discharge the supernatant.

3. Add 100 μl of Solution 1, uniformly suspend cells by pipetting.

4. Wait 5 min. At this stage pH is changed and cell membrane is destabilised

5. Add 150 μl of Solution 2, mix by inverting the eppendorf 5 times.

6. Wait 3 minutes. At this stage cells are lysed by SDS and NaOH denatures the DNA. Solution should become more transparent.

7. Add 150 μl of Solution 3, mix by inverting the eppendorf 5 times.

8. Wait 5 min. Plasmid DNA renaturates. Contaminant should be visible as a white layer.

9. Centrifuge at the maximum speed for 10 min in 4°C. The plasmid DNA is in the supernatant. Contaminants are in the pellet

10. Pippete the supernatant into the new eppendorfs.Be careful not to pipette the pellet.

11. Add 0,8 ml of the pure isopropanol into each eppendorf mix gently. It precipitates the plasmid.

12. Incubate at -20 or -80 for 10 min or more. Improves efficiency.

13. Centrifuge at the maximum speed for 10 min in 4°C.

14. Discharge the supernatant. Plasmid is in the pellet.

15. Add 1 ml of 70% ethanol into each eppendorf to rinse the pellet.

16. Centrifuge at the maximum speed for 10 min in 4°C.

17. Discharge the supernatant gently and air-dry the eppendorfs for 10-30 minutes to allow the ethanol to evaporate.

18. Suspend the dry pellet in 40 μl of Tris-EDTA buffer with RNAses.

Procedure
1. Take the eppendorf with chemically competent cells out of -80°C.

2. Defrost on ice for 10 minutes.

2. Add the DNA (10-20 μl if cells are transformed with ligation mix / 4 μl of the DNA if cells are transformed with the DNA from 2009 distribution kit plates).

3. Gently mix by pipetting the cells.

4. Chill on ice for 30 minutes.

5. Heat shock at 42°C for 60 seconds.

6. Chill on ice for 10 minutes.

7. Add 1mil of LB broth.

8. Incubate at 37°C for 1 hour.

9. Plate 100μl of the culture on the agar plate with appropriate antibiotic.

10. Centrifuge at 6 000 rpm for 2 min.

11. Discharge most of the supernatant. Leave around 100 μl total volume.

12. Suspend the pellet in the reminded supernatant.

13. Plate 100μl of the concentrated culture on the agar plate with appropriate antibiotic.

14. After transformation cells need to be grown under selection conditions.

Selective media preparation
0,1g/ml Ampicilin and Kanamycin solutions are stored in the freezer. Agar plates with appropriate antibiotic should be fresh to maintain the proper concentration of the antibiotic.
 * LB-Agar-Ampicilin plates. Into 200 ml of cold Agar and LB add 200 μl of 0,1g/ml Ampicilin solution.
 * LB-Agar-Kanamycin plates. Into 200 ml of Agar and LB add 200 μl of 0,1g/ml Kanamycin solution

Spectrophotometry
We use Nanodrop ND-1000 to measure the DNA concentration in the 1ul sample. The apparatus is always cleaned and calibrated before use.

Gel electrophoresis
TBE buffer is usually used, it is best for resolution of 0.1-3kb fragments, TAE is better for fragments>4kb. In general smaller molecules travel faster, but nicked or open circular plasmid will move slower than linearised plasmid of the same size and linearised is slower than supercoiled. The concentration of agarose affects the resolution, 0.8% gel is usually used. Chose the gel concentration according to following table: 1. Agarose and TBE are mixed in the appropriate amounts.

2. The mixture is heated in the microwave and mixed until agarose fully dissolves.

3. Cool the beaker with the mixture in the cold water until it no longer burns your hands.

5. Insert the comb, pour the gel into the gel tray. Gel should be thin, but thick enough to form wells.

6. Keep at room temperature until it solidifies.

7. Keep in the refrigerator for 5-7 min.

8. Place the gel tray into the running bay.

9. Add TBE buffer to  cover the gel.

10. To every DNA sample add the loading dye, around 2μl per 20μl. Use the dye *without* SDS and EDTA if you want to extract the DNA from the gel.

11. Load the samples and 2μl of the DNA ladder.

12. Switch on the apparatus, set the voltage to 80-100V

13. Check the current intensity.

14. Wait until ready (around 1.5h)

15. Place the gel in the EtBr solution for 10 mins.

16. Place the gel in the UV visualiser.