Purification of nucleic acid

Plasmids are the most common vectors used in genetics and biotechnology labs.

A foreign DNA (a particular gene) can be inserted into a plasmid vector and expressed in competent bacterial cells in the presence of a selective antibiotic marker. Plasmids are also used for producing large amounts of protein from the inserted gene in appropriate host cells such as bacteria or yeast [Gupka, 2008].

26

To investigate the expression and the role of different proteins in melanoma cells, both DNA and RNA were isolated from cells and used for different experiments in this study.

3.1.1 Protocol for plasmid purification with Nucleobond®Xtra Midi Kit

Nucleobond®Xtra Midi Kit from Machery- Nagel (Table 2.1) was used to purify high-copy plasmids from Escherichia coli (E. coli), DH5 strain bacterial culture. The method consists on ionic interaction of positively charged silica-based membrane with negatively charged phosphate backbone of the plasmid DNA, binding of nucleic acid to the column, followed by the elution of pure DNA in alkaline conditions [user manual 2012 including references available at

http://www.mnnet.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20P urification/UM_pDNA_NuBoXtra.pdf].

Bacteria transformed with the plasmid of interest were grown overnight in 100ml LB containing the appropriate antibiotics (Table 2.10) at 37°C and 220 rpm. The cells were harvested by centrifugation at 6000 rpm for 10 minutes at 4°C, then the supernatant was discarded and the pellet resuspended in 8 ml Resuspension buffer (RES) containing RNase to eliminate all the RNA. Cells were thereafter lysed through alkaline lysis with NaOH/SDS by adding 8 ml of lysis buffer (LYS) and inverting the tubes 4-6 times before incubation for 5min at RT. NaOH breaks the cell wall and disrupts the hydrogen bonding between DNA bases, converting dsDNA into ssDNA and SDS denatures most of the proteins in the cell, helping the separation of the proteins from the plasmid later in the process. During the incubation time, the column and the inserted column filter were equilibrated by applying 12 ml equilibration buffer (EQU) to the rim of the column filter and allowing the column to empty itself by gravity flow. The lysate was neutralized by adding 8 ml neutralization buffer (NEU) and mixing by gentle inversion of the tubes 10 to 15 times before loading onto the filter. Neutralization solution contains potassium acetate which decreases the alkalinity of the mixture, allowing the plasmid DNA to re-nature to dsDNA, while genomic DNA, SDS and denatured cellular proteins stick together and form a white precipitate.

The lysate was then poured on the column filter and loaded into the column. When column had emptied, the filter was removed and the column was washed with 5 ml equilibration buffer (EQU) and 5 ml of washing buffer (WASH) and plasmid DNA was eluted from the column by adding 5 ml elution buffer (ELU). The eluted DNA was precipitated by adding 3.5 ml isopropanol at room temperature. The DNA was pelleted by centrifugation at 15,000 x g for 30

27

minutes at 4°C, subsequently washed with 5 ml of 96% ethanol at room temperature. After a quick centrifugation at 15,000 x g for 5 minutes at 4°C, the ethanol was discarded and pelleted plasmid DNA was resuspended in 200 µl 1x TE buffer. The DNA concentration and purity was measured by Nanodrop.

3.1.2 Protocol for purification of DNA from cultured cells

QIAmp DNA mini kit from Qiagen (Table 2.1) was used to isolate DNA from melanoma cells.

Large flasks (T175) were used for growing the cells until 90% confluency. Cells were then enzymatically detached by using trypsin, counted and up to 5x10⁶ cells were pelleted by centrifugation for 5 min at 200xg.The cell pellet was resuspended in an Eppendorf tube with 200µl PBS, then lysed by adding 200µl AL buffer and proteins denatured by adding 20µl proteinase K. Samples were mixed by pulse-vortexing for 15 sec. and incubated for 10 min at 56⁰C for an efficient lysis. In the next step, 200µl ethanol 96% was added to the samples, followed by a brief mix by pulse-vortexing for 15 sec. The lysate was transferred to a spin column in a 2ml collection tube and centrifuged for 1min at 6000xg.The spin column was washed with 500µl washing buffer AW1 and AW2, the filtrate discarded each time in a new 2ml collection tube by centrifugation for 1 min at 600xg and 20.000xg for 3 min, respectively. After removing the contaminants, DNA was eluted in 200µl elution buffer (AE), incubated for 5 min at RT then centrifuged at 6000xg for 1min.The concentration and the purity of the DNA were measured with Nanodrop. The samples were stored at -20⁰C until use.

3.1.3 Protocol for purification of total RNA

To isolate RNA from mammalian cells the Nucleospin®RNAII kit from Machery-Nagel was used [user manual 2011 and references available at

http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/RNA%20and%20mRNA/UM_TotalR NA.pdf].

The protocol allows the purification of up to 70µg of total RNA from up to 5x10⁶ eukaryotic cells. All steps were performed at room temperature and all centrifugation steps were performed at 11,000xg. The cells of interest were grown in T125cm² flask, collected in a 15ml tube by centrifugation and lysed by addition of 350µ of RA1 buffer and 3,5µl 1M DTT. The RA1 buffer contains large amounts of chaotropic ions, thus preventing the degradation of RNA by

28

inactivating RNases and creating appropriate binding conditions for adsorption of RNA to the silica membrane. The lysate was filtrated by centrifugation for 1 minute. After the removal of the filter, 350µl of 70% ethanol was added for adjusting RNA binding conditions. The lysate was loaded into a column and centrifuged for 30 seconds, the silica membrane desalted with 350µl MDB (Membrane desalting buffer). Salt removal improves the digestion of contaminating DNA by rDNase solution (10µlrDNase and 90µlReacton Buffer for rDNAse) which is directly applied to the silica membrane followed by a 15minutes incubation time. Salt, metabolites,

macromolecular cellular components were removed from the silica membrane with 2 different washing buffers (200µRA2 ,600 and 250µl RA3buffer) in 3 washing steps, each step followed by centrifugation for 30 seconds ( 2 minutes in the last step ). Pure RNA was finally eluted under low ionic strength conditions with RNase-free water. The concentration and quality of total RNA was determined by NanoDrop and immediately placed at -70⁰C to avoid its degradation.