2.2.1.2.4.1 SMART first strand cDNA synthesis protocol
2.2 A.2 Transformation of competent cells
2.2.4 A Plasmid purification
2.2.4.4.1 Miniprep
M iniprep plasm id DNA purifications were perform ed to check correct insertion of ligations into the chosen vector by restriction digest. For this purpose the Q iagen spin m iniprep kit was employed. This kit is based upon the alkaline lysis o f bacterial cells (Birnboim and Doly, 1979) followed by adsorption o f D N A onto silica in the presence o f high salt. RNA, proteins, and low -m olecular-w eight im purities are rem oved by a m edium -salt wash. Plasm id DNA is eluted in a low -salt buffer, and then concentrated and desalted by isopropanol precipitation. The buffers supplied with kit are sum m arised in Table 2.9. 1.5 ml o f sm all-scale culture was centrifuged at 12,000 x g to pellet the
E.coli. Supernatant was rem oved and the cells re-suspended in 250 p,l o f cell suspension buffer P I, containing RN ase A. 250 pi cell lysis solution P2 was added to the hom ogenous cell suspension and the tubes w ere incubated at room tem perature for 5 min. This allowed sodium dodecyl sulphate (SDS) to denature the bacterial proteins, N aO H to denature the chrom osom al and plasm id DNA and RN ase A to digest the RNA. 350 pi high-salt neutralisation buffer N3 was then added to each tube, w hich was capped and inverted 4-5 times to mix the contents. V ortexing was avoided at this step to prevent shearing o f genom ic DNA. The suspension was centrifuged 12,000 x g for 10 min at room tem perature and the supernatant was transferred to a spin-cartridge placed in a wash tube. The spin-cartridge/wash tube assem bly was centrifuged at 12,000
X g for 1 min at room tem perature and the flow -through was discarded. The spin-
cartridge was then w ashed with 700 pi o f wash buffer PE, and centrifuged at 12,000 x g for 1 min at room temperature. The spin-cartridge was then placed in a 1.5 ml recovery tube and 75 pi of w arm TE buffer was added directly to the centre o f the spin-cartridge. A fter an incubation period of 1 min the spin-cartridge/recovery tube was centrifuged at
C hapter 2 M aterials & M ethods Buffer Composition PI Cell Suspension Buffer (stored at 4°C) 50 mM Tris-HCl (pH 8) 10 mM EDTA, RNase A lOOpg/ml
P2 Cell Lysis
Solution 200 mM NaOH, 1% SDS (w/v)
N3 Neutralisation
Buffer
Proprietary high salt buffer to create dehydrating conditions required for DNA to bind silica in miniprep columns. Contains 25-50% guanidinium
chloride and 10-25% acetic acid
P3 Neutralisation
Buffer
3.0 M Potassium Acetate pH5.5, Guanidine hydrochloride
PE Wash Buffer Proprietary Ethanol based wash buffer
QBT Equilibration
buffer
750 mM NaCl, 50 mM MOPS pHV.O, 15%, isoproanol, 0.15% Triton X-100
QC Wash Buffer 1.0 M NaCl, 50mM MOPS pH 7.0,
15% isopropanol
QF Elution buffer 1.25 M NaCl, 50mM Tris Cl pH 8.5,
15% isopropanol
Table 2.9 - Plasmid purifîcation kit buffer compositions
2.2.4.4.2 Maxi-prep
The Q iagen m axiprep kits were used in large-scale plasm id purifications. These kits are based upon the alkaline-lysis technique followed by binding o f plasm id D N A to an anion-exchange resin under appropriate low -salt and pH conditions. All the necessary buffers are essentially as described in Table 2.9. 200-250m l of large-scale bacterial culture was centrifuged at 6,000 x g for 15 m in at 4°C to pellet the bacteria and the supernatant was removed. The cell pellet was re-suspended in 10 ml o f B uffer P I. 10 ml of buffer P2 was then added to lyse the bacteria. 10 ml o f chilled buffer P3 was added to neutralise and term inate these reactions. The suspension was centrifuged im m ediately at 20,000 X g for 30 min at 4°C then the supernatant was re-centrifuged for a further 15
min to rem ove any rem aining particles. The supernatant was poured onto an equilibrated anion-exchange colum n (15ml QBT) that binds double and single stranded D NA , and RN A and allowed to drain by gravity flow. The nucleic acids w ere then w ashed w ith 60 ml o f W ash B uffer QC. The plasm ids were eluted from the colum n by adding 15 m l elution buffer QF. 10.5 ml isopropanol was added to the eluate and the m ixture was centrifuged at 15,000 x g for 30 min at 4°C in order to precipitate and pellet the DNA. The supernatant was discarded and the D N A pellet was w ashed with 5 ml 70% ethanol and re-centrifuged at 15,000 x g for 10 min at room tem perature. The
C hapter 2__________________________________________________M aterials & M ethods
pellet was air-dried and dissolved in the appropriate am ount o f water. The DNA concentration was checked by using a Beckm an spectrophotom eter and diluted to a w orking concentration of Ijig/fil.