Construction Of Genomic Lambda Libraries

The source of DNA for the first lambda library constructed was total EcoRI digested DNA, from a homozygous insertional mutant mouse, which had been size-selected for fragments > 4kb on a sucrose gradient. This was performed by Dr. Susan Darling. Two further libraries were constructed using genomic DNA isolated from mouse liver; the first was a homozygous insertional mutant mouse (Tg) and the second a wild-type [C57BL/6JxC3HeB/FeJLe-a] mouse (wt).

2.6.1 Partial Digestion Of Genomic DNA

The latter two libraries were constructed using the genomic replacement lambda phage vector, BamHI, which is capable of accepting BamHI compatible fragments (Mbol, Sau3AI, Bglll, N dell or BamHI) ranging in size from 9 to 23 kb. Pilot digests were set up on both DNA samples using N dell to ascertain the optimal conditions for obtaining DNA fragments in this size range. The following mix was set up for each DNA sample:

30fig DNA made up to 900p.l with TE pH 8 lOOp.1 lOX restriction enzyme buffer

These were stored at 4°C for ~2 hrs. Ten 1.5ml tubes were labelled, 1 to 10, for each sample. In tube 1, 60(il of the above mix was aliquoted and in tubes 2 to 10 30p.l of the mix was aliquoted, all manipulations were perfonned on ice. Two units of N dell was added to tube 1 and mixed gently with a pipette tip. 30ql of tube 1 was transferred to tube 2 and mixed gently. This transfer of 30|il was repeated for tubes 2 to 9. Nothing was added to tube 10 and 30|il was discarded from tube 9. All reactions were incubated at 37°C for 1 hr, stopped by the addition of EDTA to a final concentration of 5mM and stored at 4°C. The pilot partial digests were run on 0.4% agarose gels supported on a 1% agarose gel, with X H indlll as a size marker, for 4 hrs at 50V. For both DNA samples, the conditions in tubes 7, 8 and 9 gave fragment sizes that were mainly between 9 and 23 kb.

Large scale digests were set up using the conditions used for tubes 7, 8 and 9. A total of 150|utg of DNA from each sample was digested at each specification, so that in total, 450|ig of DNA from the wild-type and insertional mutant mice was

digested. The reaction conditions for tubes 7, 8 and 9 are given in Table 5. Five tubes were set up, each containing 30|ig of DNA, for each specific reaction

condition. The DNA, restriction buffer and TE pH 8 were mixed together and stored at 4°C for ~2 hrs, the appropriate amount of enzyme was added and the reactions incubated at 37°C for 1 hr. The digests were stopped by the addition of EDTA to a final concentration of 5mM and stored at 4°C. Aliquots of the digests (~ 0.5|ig) were run on a 0.4% agarose gel to check that they had been suitably digested. The 5 separate partial digests containing 30|Ltg of DNA were combined in 1 tube to give 150|ig of partially digested DNA, i.e. wt7; wt8; wt9; Tg7; Tg8; Tg9. To each tube an equal volume of P:C:I was added, mixed gently and centrifuged at 13000 rpm for 5 mins. The upper layer was transferred to a clean tube and a second P:C:I

extraction performed. The cleaned upper layer was extracted with an equal volume of chloroformiisoamyl alcohol, mixed and centrifuged at 13000 rpm for 5 mins. The upper layer was transferred to a clean tube, 0.1 volumes of 3M sodium acetate pH 5.2 and 2 volumes of 100% ethanol were added, mixed and incubated at -20°C for 30 mins. The samples were centrifuged at 13000 rpm for 5 mins, the DNA washed in 70% ethanol, air dried and resuspended in 300fil of TE pH 8 at 4°C overnight.

2.6.2 Size Selection Of Genomic DNA Using Sucrose Gradients

The partial Ndell digested DNA from the wild-type and insertional mutant mice was size selected to enrich for fragments > 6kb; this was done by running the samples through a sucrose gradient. The sucrose gradient started at 20% and finished at 45%, increasing in 5% graduations. Sucrose solutions were made up in IM NaCl; 0.02M Tris-HCl; O.OIM EDTA pH 7.5 at 20%, 25%, 30%, 35%, 40% and 45% concentrations. The gradients were set up in 13ml ultracentrifuge tubes, 1.5ml of the 20% sucrose solution was added first, using a syringe and needle, followed by the 25%, 30% up to the 45%, always injecting the solution into the bottom of the tube, so that the 20% solution was the top layer and the 45% solution the bottom. The finished sucrose gradients were covered with Nesco film and stored at -70°C

overnight. The sucrose gradients were thawed for 15-20 mins and the partial digests loaded on top; 6 gradients were set up and loaded with wt7, wt8, wt9, Tg7, Tg8 and Tg9, 300ql of DNA for each. The gradients were overlaid with mineral oil, to within 2-3mm of the top of the tubes and loaded into the buckets of the SW41 rotor in the

Table 5. Reaction Conditions For N d ell Partial Digests On Mouse Genomic DNA

Tube Number Total Volume (111) Amount Of DNA (lig) Amount Of N d e ll (units) 1 30 0.9 1 2 30 0.9 0.5 3 30 0.9 0.25 4 30 0.9 0.125 5 30 0.9 0.0625 6 30 0.9 0.03125 f 30 0.9 0.015625 8* 30 0.9 0.078125 9^ 30 0.9 0.0039062 10^ 30 0.9 0

* The reaction conditions in tubes 7, 8 and 9 were used to set up large scale partial N dell digests on genomic DNA from the wild-type and insertional mutant mice.

Beckman Optima L ultracentrifuge. The tubes were run at 26000 rpm for 20 hrs at 20°C with no brake. The tubes were removed from the rotor, very carefully, and 500|il fractions were isolated by piercing the bottom of the tube with a needle and catching the drops; approximately 20-22 fractions were collected from each tube and stored at 4°C. To ascertain which fractions contained the DNA of the required size,

lOp-1 of every other fraction was mixed with lOjLil of water and 5|il loading buffer. These were then run on 0.4% agarose gels, poured on 1% support gels. The X H indlll marker was made up in sucrose to a final concentration of 30%, as this was the sucrose concentration of the fractions isolated from the gradients, and the

presence of sucrose affects the mobility of DNA. The gels were run overnight at 25 V and visualised under UV light. Fractions 1-10 were selected in all 6 gradients, they all contained DNA > 6kb.

DNA was isolated by combining fractions, 1 with 2, 3 with 4, 5 with 6, 7 with 8, and 9 with 10, which gave five 1ml samples of 30% sucrose containing DNA for each sample. To reduce the sucrose concentration to below 10%, 2ml of TE pH 8 was added. A one-tenth volume of 3M sodium acetate pH 5.2 and 2 volumes of 100% ethanol were added, mixed and the DNA precipitated at -20°C for ~ 4 hrs. This was centrifuged at 10000 rpm for 20 mins at 4°C and the DNA air dried and resuspended in 100|il TE pH 8 at 4°C. DNA from every tube was run on 0.4% gels, supported on

I % gels, to check the integrity of the DNA fractions. All of the samples were intact and DNA from the 5 associated tubes (e.g. wt7, tubes 1-5) was combined and reprecipitated with 0.1 volumes 3M sodium acetate pH 5.2 and 2 volumes 100% ethanol. DNA was resuspended in 20|ll1 TE pH 8 to give 6 samples, wt7, wt8, wt9, Tg7, Tg8 and Tg9. These samples were then further combined, wt7, wt8 and wt9 together and Tg7, Tg8 and Tg9 together. This gave 2 samples of partial Ndell digested DNA, one from the wild-type (wt) and one from the insertional mutation (Tg) mouse, both having a final volume of 60|il.

The concentration of the DNA was estimated by comparison to DNA of known concentration. A petii-dish containing 0.8% agarose and ethidium bromide, at a final concentration of 0.2}ig/ml, was poured and allowed to set. One microlitre of the Nde II digested DNAs, wt and Tg, and the test insert, pME at a concentration of

viewed under UV light and the relative intensities of the spots compared. Both the wt and Tg DNAs showed a similar intensity as the standard s o l estimated that both DNAs were at approximately 0.25|Lig/|il.

2.6.3 Ligations