Team:TU Munich/lab/notebook/methods

Restriction digest

1. Add 1ug of DNA to be digested, and adjust with dH20 for a total volume of 43ul.

2. Add 5ul of NEBuffer 4 to the tube.

3. Add 1ul of your first enzyme.

4. Add 1ul of your second enzyme.

5. There should be a total volume of 50ul. Mix well and spin down.

6. Incubate the restriction digest at 37C for 60min, and then 80C for 20min to heat kill the enzymes.

7. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 10ul of the digest (200ng of DNA) for ligations.

Quick Ligation Protocol Quick T4 DNA Ligase = Quick Ligase M2200L

Buffers: Quick Ligation Reaction Buffer 2x

Protocol

1. Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 μl with dH2O.

2. Add 10 μl of 2X Quick Ligation Buffer and mix.

3. Add 1μl of Quick T4 DNA Ligase and mix thoroughly.

4. Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.

5. Chill on ice, then transform or store at -20°C.

Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.

Ligation (as of june 27th 2011)

1. 6 µl of ddH2O

2. 10 µl of each sample you will be ligating (destination plasmid and part)

3. 3 µl of T4DNA Ligase Reaction Buffer

4. 1µl of Quick DNA Ligase

5. Mix well and spin down

6. Incubate for 60 min at 20°C and 20 min at 80 °C to heat kill

7. use 1 µl of ligation to transform into competent cells

Transformation

1 µl of desired plasmid (better: 0.5 µl in order to avoid arcing) was added to a tube containing 40 µl electrocompetent cells. The solution was gently mixed while steering with pipette tip. The mixture was transferred into a chilled cuvette (1 mm gap) and immediately inserted in the electroporation device. Electroporation was performed using 1510V. Directly after electroporation, 1 ml of room temperature SOC-medium was added per cuvette. The solution was mixed and transferred into 1ml Eppendorf tube for incubation at 37 °C, 200-300 rpm and 1-2 h. Afterwards, 100 µl and 100 µl concentrated (spun down and resuspended in 400ul) of each transformation reaction was spread on LB plates containing the appropriate antibiotics and raised over night at 37 °C. On the next day, colonies were picked and inoculated into 5 ml LB medium supplied with the appropriate antibiotics. This is either done VERY early in the morning, or late in the evening (in order to continue the following day). Next, the plasmid can be prepped.

Preparing electro competent cells

Cell culture: A glycerol stock culture was spread on LB/agar plates and incubated over night at 37 °C. The following day, a single colony was picked and transferred into 20 ml LB-Luria media, which was pre incubated at 37 °C and 250 rpm in a 250 ml Erlenmeyer flask. The cells were cultivated over night during shaking at 37 °C. On the next day, 300 ml LB-Luria in 1 L Erlenmeyer flask was raised to 37 °C and inoculated with 5 ml of over night culture. Incubation was carried out (37 °C, 300 rpm) until the OD was measured between 0.4 – 0.5. Cell culture desalting: To increase the resistance of the sample solution and therefore prevent arcing during electroporation and high voltage, salts were washed out from cell suspension. Cells with an OD of 0.444 were immediately chilled on ice for 15 min and 5x about 50 ml were transferred into pre chilled falcon tubes. Harvesting of cells was performed at 2.000 rcf and 4 °C for 20 min . Each pellet was re-suspended in 5 ml sterilized and pre-chilled H2O and again centrifuged for 10 min at 4 °C and 1.500 rcf . In the following step, cells were washed twice with á 50 ml 10% Glycerin and centrifuged with 1.500 rcf for 20 min. Then 20ml with 1500 rcf for 10min. Then 5ml 1500 rcf for 10 min. After the washing procedure, cells were re-suspended in an end volume of 1 ml and merged. The optical density was determined. As described in protocol 26 of Sambrook and Russell an OD value of 1 is equal to 2.5 x 10^8 cells/ml. The final concentration should be ca. 10^10 per ml (?).

[edit] Miniprep (Metabion mi-plasmid mini prep kit)

ADD RNase TO "CELL RESUSPENSION SOLUTION" AND 100% EtOH TO "COLUMN WASH BUFFER"!!!

All centrifugation steps are performed at 13.000 rpm and RT!

1. Take 1.5 to 2 ml (max. 5 ml) bacterial overnight LB culture

2. Centrifuge for 1 min

3. Discard the supernatant completely (Removing all liquid at this step is critical!)

4. Add 250 μl of Cell Resuspension Solution and resuspend the pellet completely by vigorously vortexing

5. Add 250 μl of Cell Lysis Buffer to the cell suspension (If the Lysis Buffer stock solution is precipitated, heat to 55°C for 5 min to dissolve -> RT)

6. Invert 5 times to mix. Do not vortex, as this will cause chromosomal DNA contamination of the plasmids! Incubation < 5 min (usually 3 min)

7. Add 350 μl of DNA Binding Buffer

8. Invert 5 times to mix (Do not vortex, see step 6)

9. Centrifuge for 10 min

10. Transfer all of the clear liquid supernatant to a spin column which has been set into a 2 ml collection tube

11. Centrifuge for 1 min

12. Add 600 μl of Column Wash Buffer to the spin column

13. Centrifuge for 1 min

14. Repeat steps 12 and 13 to make sure, no Column Wash Buffer is left in the column

15. Place the spin column in a new 1.5 ml Eppi

16. Elute the plasmid DNA by adding 50 μl of autoclaved water directly onto the middle of the white membrane of the spin column. Incubate for 1 min.

17. Centrifuge for 1 min

18. Directly use or store at -20°C

Preparing electro competent cells

Cell culture: A glycerol stock culture was spread on LB/agar plates and incubated over night at 37 °C. The following day, a single colony was picked and transferred into 20 ml LB-Luria media, which was pre incubated at 37 °C and 250 rpm in a 250 ml Erlenmeyer flask. The cells were cultivated over night during shaking at 37 °C. On the next day, 300 ml LB-Luria in 1 L Erlenmeyer flask was raised to 37 °C and inoculated with 5 ml of over night culture. Incubation was carried out (37 °C, 300 rpm) until the OD was measured between 0.4 – 0.5. Cell culture desalting: To increase the resistance of the sample solution and therefore prevent arcing during electroporation and high voltage, salts were washed out from cell suspension. Cells with an OD of 0.444 were immediately chilled on ice for 15 min and 5x about 50 ml were transferred into pre chilled falcon tubes. Harvesting of cells was performed at 2.000 rcf and 4 °C for 20 min . Each pellet was re-suspended in 5 ml sterilized and pre-chilled H2O and again centrifuged for 10 min at 4 °C and 1.500 rcf . In the following step, cells were washed twice with á 50 ml 10% Glycerin and centrifuged with 1.500 rcf for 20 min. Then 20ml with 1500 rcf for 10min. Then 5ml 1500 rcf for 10 min. After the washing procedure, cells were re-suspended in an end volume of 1 ml and merged. The optical density was determined. As described in protocol 26 of Sambrook and Russell an OD value of 1 is equal to 2.5 x 10^8 cells/ml. The final concentration should be ca. 10^10 per ml (?).

Glycerin Stocks

A mixture of 50 % LB and 50 % Glycerin is prepared and autoclaved. 300 ul of this solution are added to 700 ul cells, in order to get a about 10 - 15 % glycerin solution supplied with the cells to be frozen. The solution is then frozen at -80 °C.

S-gal plates

for 500 ml:

0,5l Milli Q water

7,5 g AgarAgar

10g LB-Pulvermix

460 mg ferric ammonium sulfate (do NOT dissolve s-gal just add the powder150 mg S-Gal )

100 microgramm/ml Antibiotics

put everything together and autoclave

normally 250 mg of ferric ammonium citrate (261,97 g/ml) are used here it is replaced by ferric ammounium sulfate (482,25 g/mol)

you don't need to keep s-gal out of the light it is heat and light-INSENSITIVE

do not store prepared s-gal plates longer than 14 days!

Squeeze N Freeze

1. Electrophorese the DNA sample in an agarose gel (TAE or TBE), then stain with an appropriate reagent, e.g., ethidium bromide or SYBRTM Green I.

2. Using a clean razor blade, carefully excise the band of interest. Trim excess agarose from all six sides of the DNA band to maximize recovery and purity.

3. Chop the trimmed gel slice and place the pieces into the filter cup of the Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column. Place the filter cup into the dolphin tube. If the volume of your trimmed gel slice is too great to fit into one filter cup, then use two or more and pool the recovered samples at the end of the protocol.

4. Place the Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column (filter cup nested within dolphin tube) in a -20° C freezer for 5 minutes.

5. Spin the sample at 13,000 x g for 3 minutes at room temperature.

6. Collect the purified DNA from the collection tube; the agarose debris will be retained within the filter cup of the Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column. The DNA is ready to use for PCR, ligations, labeling or other enzymatic reactions. Ethanol precipitation is recommended for applications requiring a more concentrated sample and will also have the effect of further purifying the sample.

Nucleic Acid Precipitation from Diluted Solutions with Glycogen Protocol from http://www.fermentas.com/en/products/all/reagents/r056-glycogen

1. Add 1/10 volume of 3 M Sodium Acetate (or 2 M sodium chloride, or 5 M ammonium acetate) to DNA solution.

2. Add Glycogen (#R0561 for DNA or #R0551 for RNA) to a final concentration of 0.05-1 µg/µl. Use up to 1 µl of Glycogen per 20 µl of solution.

3. Add 2.5 volumes of ethanol. Mix gently but thoroughly.

4. Incubate for 5 min at room temperature.

5. Centrifuge the mixture for 10-15 min at 10,000 rpm. Discard the supernatant.

6. Rinse the pellet with cold 70% ethanol. Air-dry the pellet.

DNA: Dissolve the pellet in Water, nuclease-free or TE buffer.

RNA: Dissolve pellet in DEPC-treated Water.

Glycogen/ethanol precipitation

1. add 2 µl 3 M sodium acetate, 0.5 µl glycogen and 22.5 µl isopropanol to 20 µl ligation product

2. incubate the mixture at -20°C for one hour

3. centrifuge the mixture for 15 minutes at 18,000 rpm

4. discard the supernatant

5. add 50 µl 70 % ethanol

6. centrifuge the mixture for another 10 minutes at 18,000 rpm

7. remove supernatant with a pipet CAREFULLY. If pellet moves, centrifuge again.

8. air-dry the pellet until the ethanol is completely evaporated. Do not over-dry pellet as this makes dissolving more difficult.

9. resuspend the pellet in 10 µl nuclease-free water by pipetting carefully.

subsequent transformation of purified ligations:

10 µl purified ligation was mixed with 40 µl competent cells and electroporated into DH5alpha. 1 ml SOC medium (SOB medium + 20 mM glucose) was used and incubation was carried out for 1h at 37°C, shaking at 500rpm in 2 ml eppis. Culture was then centrifuged at 4000 rpm for 5 minutes, and resolved in 100 µl medium. Whole sample was plated onto LB/agar plates supplemented with appropriate antibiotica.

Transforming Chemically Competent Cells

Based on http://openwetware.org/wiki/Transforming_chemically_competent_cells

1. Thaw chemically competent cells on ice.
2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
   • Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
3. Let sit for 30 minutes on ice.
4. Incubate cells for 30 seconds at <math>42^o</math>C.
5. Incubate cells on ice for 2 min.
6. Add 1 mL SOC at room temp.
7. Incubate for 1 hour at <math>37^o</math>C on shaker.
   • Note: Can save some time here by reducing incubation to ~45 min.
8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
9. Grow overnight at 37 °C.
10. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.