DNA Modification reactions

1.6.1 Restriction of DNA with bacterial endonucleases tvpe II

The endonucleases used were purchased from either GIBCO-BRL or New England Biolabs and used according to the suppliers protocols.

1.6.1.1 Restriction of low molecular weight DNA

Plasmid DNA or DNA produced by the PCR were restricted in 20 pi reaction volumes using 5-10 units of enzyme at the appropriate reaction temperature. Plasmid DNA was cleaved for 2 and 8 hours for pGEM-T vector and pBluescript respectively. If the plasmid DNA was to be cleaved with two restriction enzymes simultaneously, GIBCO-BRL buffer 3 was used in the presence of 10 mM of DDT. Bacteriophage X DNA was restricted in 100 pi reaction volume in the presence of 20-30 units of the appropriate enzyme and always using the One-Phor-All Buffer Plus (Pharmacia) for 5

hours. Combinations of two restriction enzymes were also used in this buffer. For PGR- generated DNA, two hours of digestion were sufficient. Mouse and yeast genomic DNA was restricted in 40 fxl reaction volumes in the presence of 20-30 units of restriction enzyme. Restricted yeast DNA, destined for vectorette library construction, was precipitated by adding 2 volumes of 99.9% ethanol in the presence of 0.3M of NaOAc, after heat-inactivating the restriction enzymes (65°C or 75°C for 10 minutes). The DNA was pelleted by centrifugation at 13000 g for 15 minutes, washed in 70% ethanol, dried by heating to 75°C for 2 minutes, and resuspended in the appropriate volume of ddH20.

1.6.1.2 Restriction of high molecular weight DNA embedded in agarose blocks

The agarose blocks were washed four times (30 minutes each time) in excess of ddH20 and placed in a dry plate. Excess water from the blocks was removed by aspiration, using a Gilson micropipette. A single block has a 100 pi displacement volume. The reactions were set up in 200 pi volume, containing the appropriate buffer and 40-50 units of the restriction enzymes. The digestions were left overnight. For two sequential digestions of the same sample, the blocks were washed with excess water as above after the first digestion and the new restriction reaction set up. For enzymes with compatible buffers the blocks were also washed. The second digestions were again left overnight.

1.6.2 Déphosphorylation of DNA

The 5’-phosphate residues from digested DNA (insert) were removed using calf intestinal phosphatase (CIP; Pharmacia) to prevent the formation of chimeric DNA fragments. The DNA was digested in One-Phor-All Buffer Plus. The restriction enzyme was first inactivated by heating to either 65°C or 75°C (depending on the enzyme) for 10 minutes. Typically up to 5 pg of DNA was treated with 0.1 units of CIP at 37°C for 1 hour. The enzyme was inactivated by heating to 85°C for 15 minutes and the mixture maintained at room temperature for about one hour to allow reannealing of any domains that became partially denatured (especially the ends). After this stage, the DNA was precipitated as described in section 1.6.1.1.

1.6.3 Ligation of DNA

All the ligation reactions were carried out at 14°C overnight (except for the bacteriophage library construction which was performed at 4°C; for details see section

1.6.3.1 Subcloninq of bacteriophage X DNA 1.6.3.1.1 Non -directional cloning

The enzyme used for this cloning was Sau3A I. The resulting fragments originate from both the arms of the vector and the cloned mouse DNA. Fully-restricted and CIP-treated DNA was ligated to non-CIP-treated pBluescript (cleaved with the restriction enzyme BamH I) in the presence of 1X ligation buffer and 2 units of T4 DNA ligase (Pharmacia).

1.6.3.1.2 Directional cloning

Four micrograms of bacteriophage X DNA was digested with the enzymes Sst I and Hind III (for bacteriophage X vector LambdaGEM™-11; Promega) or Sst I and Xho I (for bacteriophage X vector XFIX II; Stratagene) and precipitated after heat- inactivation of the enzyme. Five hundred nanograms of DNA was ligated to 50 ng of pBluescript, digested with the appropriate combination of two enzymes, in a 20 pi reaction volume as above. The two types of bacteriophage X vectors used {X FIX II and XGEM-11) contain a unique Sst I site at either end of the cloned DNA and do not contain either a Hind III or a Xho I site in their arms. So the restriction of the clones with Sst I and one of the latter two enzymes, gave fragments which could only originate from the insert. In this respect, this approach is more efficient for cloning only insert DNA than using Sau3A I.

1.6.3.1.3 Cloning of PCR products

All the PCR products cloned ranged in size between 400 and 700 bp. A single set of conditions was followed irrespective of the size of the DNA fragments. The vector and other reagents used are commercially available (pGEM-T Vector System I; Promega). Always three microlitres of non-purified PCR product (about 25 ng) was ligated to 50 ng of pGEM-T Vector in the presence 1X ligase buffer and 1 unit of T4 DNA ligase in a 15 pi total reaction volume. Using these set of conditions, about 80% of true recombinant plasmids were usually obtained.

2.1.7 Transformation of competent E. Coli cells

For transformation, the E. Coli strain XLI-Blue was used. Both the plasmid vectors used (Pbluescript and pGEM-T vector) carry the ampicillin resistance gene. The selection between recombinant and non-recombinant plasmids is based on the a- complementation (Ullmann et ai, 1967) of the p-galactosidase gene, commonly known as blue/white selection. The ligation mix was incubated on ice with 200 pi of competent cells for 45 minutes, heat-shocked at 42°C for 30 seconds and placed again on ice for

5 minutes. To this1 ml of pre-warmed (at 37°C) LB medium was added and incubated at 37°C for 1 hour, to allow the transformed cells time to express the ampicillin- resistance gene. A fraction of the culture (200-300 pi) was mixed with 20pl of 20 mg/ml of 5-bromo-4-chloro-3-indolyl-p-D-galactosidase (X-gal) and 4pl of 200 mg/ml isopropylthio-p-D-galactoside (IPTG) and spread onto LB-agar plates containing the 50 pg/ml ampicillin. The plates were incubated at 37°C overnight. When replica plates were to be made from the transformants, the LB agar plates were overlaid with nylon membrane (Hybond N) before spreading the culture.