DNA isolation

3.2.2.1 Mini preparation of plasmid DNA

Lysozyme solution NaOH / SDS solution 50mM glucose

Two ml of LB medium containing 200 µg/ml ampicilline were inoculated with a single bacterial colony from a LB – plate and an overnight culture was obtained. 1,5 ml of the suspension was transferred into an eppendorf tube and centrifuged for 2 minutes at 13000 rpm. The liquid phase was removed and the pellet was resuspended in 100 µl of lysozyme solution. The mixture was incubated for 5 minutes on ice, then 200 µl of NaOH / SDS solution were added and mixed gently by inversion. A viscous lysate was incubated at RT for 10 minutes and 150 µl of 3M NaAc (pH 5,2) pipetted into a tube, followed by

incubation on ice for 10 minutes. The obtained fluffy solution was centrifuged 10 minutes in microfuge on high at RT. The supernatant was transferred into a new tube. If nessesary, the DNA was purified by a phenol/chlorophorm extraction.1 ml (2,5 volumes) cold 100%

ethanol were added to the supernatant and then centrifuged 15 minutes in microfuge at 4⁰C on high. The supernatant was then aspirated off and 0,5 ml of cold 70% ethanol were added and span for 5 minutes. The liquid phase was removed and pellet was dried at 37⁰C for 30 minutes. The dry pellets were dissolved in 40 µl of TE containing 100µg /ml RNAse A and used for restriction digestion analysis.

3.2.2.2 Mini preparation of plasmid DNA (QIAGEN method)

Plasmid preparations for later preparative usage were done accordingly to protocols and using the original mini prep QIAGEN reagents (QIAGEN handbook, April 1997).

2 – 5, or 10 ml of LB medium (depending on copy number of the plasmid used for

transformation) with 200 µg /ml ampicilline were inoculated with a single E.coli bacterial colony from LB – plate and an overnight culture was obtained. The whole amount of the overnight culture was centrifuged in a Megafuge 1.0 for 5 minutes at 5000 rpm. The cell

pellets were resuspended in 250 µl of P1 buffer (4⁰C), mixed with 250 µl of buffer P2 and incubated for 5 minute at RT. After addition of 350 µl buffer P3 and mixing, a cloudy solution was centrifuged for 10 minutes at 13000 rpm at RT. The supernatant was loaded onto mini-column and centrifuged for 1 minute at 13000 rpm. The flow-through was thrown away and 750 µl of PE buffer were loaded onto column followed by a

centrifugation for 1 minute again. The flow-through was again thrown away and the column was centrifuged for 1 minute to remove traces of ethanol. To elute DNA, 50 µl of 10 mM Tris-HCl, pH=8,5 were added and the column left for 5 minutes. The column was centrifuged and the DNA concentration was determined in the eluate.

3.2.2.3 Midi preparation of plasmid DNA (QIAGEN method)

Plasmid preparations for preparative usage were done accordingly to protocols and using original midi prep QIAGEN reagents (QIAGEN handbook, April 1997).

Using a sterile toothpick, a bit of a culture frozen in 7% DMSO was transferred into 5 ml LB-ampicilline medium and incubated at 37⁰C for 6 - 8 hours.

100 or 500 ml of LB medium (depending on copy number of the plasmid used for transformation) with 200 µg /ml ampicilline were inoculated with 100 or 500 µl E.coli bacterial suspension and an overnight culture was obtained. The whole volume was centrifuged at 8000 rpm for 10 minutes in a JA-10 rotor. The pellet was resuspended in 4 ml of P1 buffer and transferred into JA-20 centrifuge tubes. 4 ml of P2 buffer were added to the suspension and mixed followed by incubation for 5 minutes at RT. The mixture was neutralised by addition of 4 ml of P3 buffer and incubated then for 20 minutes on ice. After that the lysate was centrifuged 30 minutes at 18000 rpm in JA-20 rotor at 4⁰C and a

supernatant loaded onto a type 100 column equilibrated by QBT-buffer. The plasmid DNA binds to the QIAGEN resin in these columns. The column was washed with 10 ml QC-buffer and the DNA eluted in 5 ml of QF-QC-buffer and collected in a Corex tube. The DNA was precipitated with 0,7 volumes of isopropanol and centrifuged at 18000 rpm for 30 minutes at 4⁰C. The pellet was washed with 70% ethanol twice, air-dried and resuspended in 300-500 µl of 10 mM Tris-HCl, pH 8,5. Finally, the concentration of the DNA was determined at OD 260 nm.

3.2.2.4 Maxi preparation of phage λλλλ DNA (QIAGEN method)

An E.coli LE392 culture was grown overnight in 5 ml NZYDT supplemented with 10 mM Mg2SO4 and 0,2% maltose. The culture was diluted with 45 ml of the same medium and grown for more then 1 hour upon measuring of OD 600 nm (should be below 2, one OD is 8 x 10⁸ cells). To reach a ratio of bacteria / phages of 30 / 1, 10¹⁰ cells were infected with 3,3 x 10⁸ pfu λ phages for 30 minutes at 37⁰C without shaking. The mix was added to 250 ml of NZYDT supplemented with 10 mM Mg2SO4 and 0,2% maltose and grown till complete lysis (6 - 8 hours) at 37⁰C in a shaking incubator. 5 ml of chloroform (2%) were added to the lysate and incubated for 15 minutes at 37⁰C. The lysate was span down for 15 minutes at 8000 rpm in a JA 10 rotor and the supernatant treated accordingly to QIAGEN lambda handbook. To 250 ml supernatant 400 µl of buffer L1 were added and incubated for 30 minutes at 37⁰C. During this time all bacterial RNA and chromosomal DNA are digested. 50 ml of ice-cold buffer L2 were added to the lysate and incubated on ice for 60 minutes. Buffer L2 contains PEG for precipitation of the phage particles. The mixture was centrifuged at 18000 rpm for 10 minutes and the supernatant was discarded. The pellet was resuspended in 9 ml of buffer L3 by pipetting up and down and 9 ml of buffer L4 were added. The mixture was incubated at 70⁰C for 20 minutes and then cooled on ice. Buffer L4 contains SDS which denatures phage proteins and releases the DNA. 9 ml of buffer L5 were added to the liquid and immediately but gently mixed by inverting the tube 6 times and then centrifuged for 30 minutes, 4⁰C at 18000 rpm. The supernatant was transferred promptly to a fresh tube and the step repeated for 10 minutes. The obtained clear liquid was loaded onto a QIAGEN tip 500 equilibrated by QBT buffer and allowed it to enter the resin by gravity flow. The tip was washed with 30 ml of buffer QC and the DNA was eluted in 15 ml of buffer QF. The DNA was precipitated by adding of 10,5 ml of room-temperature isopropanol and centrifuged at 18000rpm for 30 minutes at 4⁰C. The obtained pellet was washed twice with room temperature 70% ethanol and centrifuged each time for 5 minutes at 18000 rpm for 10 minutes. The supernatant was removed and the pellet air-dried for 15 minutes followed by redissolving it in 300 µl of 10 mM tris-HCl, pH 8,5.

Finally, a concentration of the DNA was determined at OD 260 nm.

3.2.2.5 Genomic DNA isolation from mouse tissues

Digestion buffer for 50 ml:

100 mM NaCl

directly before use add 0,1 mg / ml proteinase K

Mouse liver or kidney was cut into small pieces, put into a mortar filled with liquid nitrogen and grind with a pestle. Obtained powder was filled into a 15 ml tube weighted before and weight of it was determined. For every 100 mg tissue 1,2 ml of digestion buffer were added and the mixture was incubated for 12 – 18 hours at 56⁰C in a water bath. A sample was extracted twice with equal amounts of phenol / chloroform and spun for 5 minutes at 3000 rpm in a cold room. A blue pipette tip was cut off and the viscous supernatant was transferred into a new tube. Two volumes of cold 100% ethanol were added, after that DNA pellet was seen. The DNA pellet was fished out of the tube with a a a glass hook molten from a pasteur pipette and washed twice in 500µl of 70% ethanol. The pellet was air-dried for 15 minutes and dissolved in 500 µl of 10 mM Tris-HCl pH 8,5 during an incubation of 3 hours at 37⁰C. The concentration of the DNA was determined at OD 260 nm and 15 µg were used for a restriction digestion.

3.2.2.6 DNA isolation from embryonic stem cells

NET - buffer for 50 ml:

Frozen cell pellet were resuspended in 700 µl of NET-buffer using a vortex. 70 µl of proteinase K solution were added and vortexed, then 70 µl of 10% SDS were added and

vortexed again. The mixture was incubated for 3 – 4 hours at 56⁰C followed by a new addition of proteinase K solution. The obtained lysate was incubated overnight at 56⁰C, extracted with phenol / chloroform / isoamylalcohol, with chloroform / isoamylalcohol and the DNA was precipitated upon addition of 3 volumes of 100% ethanol. The DNA pellet was fished out with a glass hook molten from a pasteur pipette, washed twice with 70%

ethanol and air – dried for 15 minutes. Depending on amount of the pellet, it was

redissolved in 50 – 150 µl of 10 mM tris – HCl pH 8,5 during 3 hours at 37⁰C in a water bath. The concentration of the DNA was determined at OD 260 nm and 15 µg were used for a restriction digestion.

3.2.2.7 DNA isolation from mouse tail biopsy

Proteinase K solution 10 mg / ml in NET – buffer, store at - 20⁰C

Fresh 1 cm pieces of mouse tails were put each into a 1,5 ml eppendorf tube, 500 µl of NET-buffer were added and vortexed. 50 µl of proteinase K solution were added and vortexed, then 50 µl of 10% SDS were added and vortexed again. The mixture was incubated with vigorous shaking at 56⁰C overnight. Hairs and other insoluble rests were spun down for 5 minutes at 13000 rpm and the supernatant transferred into a new tube.

DNA was precipitated upon addition of 500 µl of isopropanol. The DNA pellet was fished out with a glass hook molten from a pasteur pipette, washed twice with 70% ethanol and air – dried for 15 minutes. The pellet was dissolved in 100 µl of 10 mM Tris – HCl pH 8,5 during 20 minutes and 1 µl or 0,6 µl used for PCR.