IV. EL PAISAJE VEGETAL DE LA MONTAÑA PALENTINA
3. El desequilibrio en las estructuras como resultado y como condicionante de la
PCR products to be sequenced were extracted from the gels, ligated into a pTAG vector and transformed into E.coli XL-1 Blue cells, in order to produce an Kcoli cell colony transformed with the pTAG vector containing the required PCR product. The K coli colonies were then used to produce an high yield of DNA for use in the sequencing reaction. In addition, the Kcoli cells could be stored in glycerol at -70°C for friture use. The methods are given below.
2.10.1 Product recovery
The 1.5 per cent Nusieve agarose gel slices containing the PCR product to be sequenced were dissolved in 500pl TE (see Appendix) by incubation at 70°C for 15 minutes or until the gel slice had fully dissolved. The PCR product DNA was extracted by addition of 200pl phenol, mixing, and centrifugation at 12000rpm for 5 minutes. 500pl of the aqueous supernatant was transferred to a new 1.5ml Eppendorf and mixed with a further 200pl phenol and again centrifuged at 12000rpm for 15 minutes. 500pl of the aqueous supernatant was then transferred to a new 1.5ml Eppendorf and mixed with 200pl chloroform, and centrifuged at 12000rpm for 15 minutes. 500pl o f the supernatant was then taken and mixed with 30pl 3M sodium acetate and 1ml absolute alcohol, frozen at -70°C for 2-3 hours, and the precipitated DNA pelleted by centrifugation at 12000rpm for 25 minutes. The DNA pellet was then washed in 70 per cent alcohol, dried in air and resuspended in 30pl TE (see Appendix).
2.10.2 DNA ligation
The isolated PCR product was ligated into the pTAG vector (R+D Systems Europe Ltd) in a lOpl reaction mix comprising approximately lOOng (as measured by spectrophotometry) of the PCR product DNA, 50ng of the pTAG vector, Ix ligation reaction buffer (Gibco, BRL), ImM ATP and 5U T4 DNA ligase. The ligation reaction was carried out at 15°C overnight.
The pTAG vector contains an ampicillin resistance gene and the regulatory sequences o f the P-galactosidase gene {lacZ) which complement the sequence of the p-galactosidase gene present in the E.coli cells themselves, such that a functional P- galactosidase gene is produced upon transformation of the E.coli cells with the pTAG vector. Neither the host-encoded or plasmid encoded fragments o f the P- galactosidase gene are active alone. The Lac^ bacteria that result from this a - complementation are easily recognised because they form blue colonies in the presence of the substrate 5-bromo-4-chloro-3-indolyl-P-D-glactoside (X-gal). However, insertion of a fragment of foreign DNA into the cloning site o f the plasmid disrupts the a-complementation and prevents the production o f an active P- galactosidase gene. Therefore, bacteria which carry a DNA insert in the vector sequence can be easily identified since they produce white colonies.
2.10.3 Competent cells
1 ml of a stock of E.coli XL 1-Blue cells (Stratagene) were made competent by addition to 50 ml of LB solution and incubation with aeration (shaken) at 37°C for
2-3 hours until the ODeoo reading was approximately 0.6. The cells were then chilled on ice, and centrifuged at 2500 rpm at 4°C for 15 minutes. The supernatant was discarded and the cell pellet resuspended in 3 ml transformation buffer (see Appendix) on ice. The cell suspension was centrifuged as before, the supernatant removed and the cells resuspended in 3 ml of transformation buffer. The competent E.coli cells were then used in the transformations described below.
2.10.4 Transformation of E.coli cells and digestion analysis of inserts
5|li1 of the ligated DNA were transformed into 200pl competent E.coli X L l- Blue cells (as above) by incubation on ice for 20 minutes, followed by 2 minutes heat shock at 42°C and then addition of 500|li1 of LB broth containing 2 per cent glucose and incubation at 37°C for 30 minutes. 200pl of the cells were then spread on X- gal/IPTG agar plates containing ampicillin (see Appendix) and incubated at 37°C overnight. Non transformed bacteria are killed by the ampicillin. White colonies, indicating an insert was present (the presence o f an insert disrupts the a - complemention between the P-galactosidase sequences present in the vector and the E.coli DNA thus preventing the P-galactosidase gene to act on the substrate, X-gal, to form blue colonies), were picked and grown overnight in 2ml LB broth (taken from a 50ml stock containing 2mg ampicillin).
To analyse the exogenous plasmid insert in the transformed E.coli cells, E.coli DNA from each cloned culture was isolated using the ‘boiling mini-prep’ protocol essentially as described by Sambrook et al (1989). 1.5ml of the E.coli cell culture
was pelleted by centrifiigation at 6000g in a 1.5ml Eppendorf tube. The supernatant was discarded and the cells resuspended in 150pl of the TELT lysis buffer (see Appendix) with 20p.l of lysozyme (lOmg/ml). The mixture was allowed to stand at room temperature for 5 minutes, incubated in boiling water for 2 minutes and then put on ice for 5 minutes. The lysate was then centrifuged at 12000g for 20 minutes. The supernatant was removed to another tube and 200pl of isopropanol added to precipitate the plasmid DNA. The mixture was then centrifuged at 12000g for 20 minutes and the supernatant discarded. The remaining pellet was washed in 70 per cent ethanol, and then allowed to dry at room temperature. The pellet was then resuspended in 30 pi of water containing 30pg/ml RNase.
In order to analyse the size of the plasmid insert and, hence, deduce whether the plasmid DNA contains the required PCR product, 8 pi of the DNA was digested with Hind III and EcoR I (as described for the digestion of PCR products in section 2.6.3). The digested DNA was then analysed on a 3 per cent agarose gel to check that the inserts were the correct size, i.e., corresponding to the PCR product under study. The E.coli cultures containing the correct PCR product inserts were preserved for later use by addition of 200pl of the cell suspension to 200pl o f LB broth containing 20 per cent glycerol. The resultant mix was kept stored at -20°C.
2.10.5 DNA sequencing
In order to sequence the PCR product under study, 3 pi of plasmid DNA containing the correct insert as determined by restriction digestion analysis of the insert
size (described above) was sequenced using an a-^^P-labelled dideoxynucleotide Thermosequenase cycle sequencing kit (Amersham). Sequencing was primed by the M l3 forward primer provided with the kit. Cycle sequencing was carried out for 40 cycles of 95°C, 30 seconds; 55°C, 30 seconds; IT C , 1 min on a PHC-II thermal cycler. 3|nl of the sequencing products were electrophoresed on a 6 per cent acrylamide, 0.3 per cent bis- acrylamide gel (Easigel, Scotlab) at 1500V. Gels were dried and exposed to film for 18 hours. The film was then developed and the DNA sequence read fi'om the film.