...matched

The controls were matched for sex, age and BMI.

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...acids

Throughout this thesis the CDNA sequence numbering of Strosberg [77] was used for the localization of nucleic acid substitutions and deletions.

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...DNA

DNA should be a fibrous white material.

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...re-purified

ratio 52#52 1.6 indicates presence of protein and/or phenolic impurities.

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...vortexing

Mix gently for large fragments of DNA to avoid shearing. These samples are mixed by repeated gentle inversions. Do not vortex.

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...interface

A small amount of aqueous layer may be lost with each extraction. Volumes are adjusted accordingly.

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...acetate

Instead of sodium acetate, 5 M NaCl can be used. Salt is necessary to form a nucleic acid precipitate  at low nucleic concentrations. Sodium acetate may be preferred over NaCl for its buffering capacity.

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...HREF="footnode.html#922">60#60

If the DNA concentration is less than 1 40#40g/ml, addition of 10 40#40g glycogen  increases recovery by facilitating precipitation.

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...appropriately

Suspension of DNA is often in TE buffer for its buffering capacity, for restriction digest usually in water.

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...PCR

The PCR process is covered by US patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffman-La Roche.

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...aquaticus

T. aquaticus YT1, a thermophilic, eubacterial microorganism capable of growth at 70psy176 - 75psy176 C, was isolated from a hot spring in Yellowstone National Park and first described in [90] 25 years ago.

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...template

After the first few cycles, virtually all of the templates have been synthesized in previous cycles and, therefore, contain the primer sequences.

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...NAME="1064"> 

1% agarose gel is good for DNA pieces that range in size from 0.6 to 3 kb. For smaller pieces of DNA (150-700 bp), 2.5 to 3% agarose was used.

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...NAME="1067"> 

Ethidium bromide is a cancerogen, handle with care!

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...TAE

For small DNA fragments, TBE buffer is used for the preparation of the gel and the buffer to sharpen nucleic acids bands.

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...wells

Approximately 10 to 15 40#40g of genomic DNA, or 20 ng per single band of cloned DNA, is readily detected with ethidium bromide staining. If too much DNA is loaded, the band will be distorted.

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...gene

it is a fragment of the E. coli lac operon, containing the regulatory region and the coding information of the first amino acids.

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...digested

see section RE.

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...electrophoresis

In general, it is useful to have approximately three to four times the molar amount of the insert  compared to the vector for optimal formation of clones.

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...units

One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1nmol of 94#94P from pyrophosphate into Nordit-adsorbable material in 20 minutes at 37psy176 C[112]. 0.01 Weiss unit is that amount of enzyme required to catalyze the ligation of 11#11 95% of 140#40g of 59#59 HindIII DNA fragments at 16psy176 C in 20 minutes.

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...cells

The DH56#6 strain presents the following genotype: F90#90, 96#9680dlacZ97#97M15, endA1, recA1, hsdR17 (r98#98, m99#99), supE44, thi-1, gyrA96, relA1, 97#97(lacZYA-argF), U169, 100#100

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...reaction

Plaques typically start to become apparent after 6 hours of incubation. The colour change due to metabolized Xgal is not apparent until 8-10 hours of incubation.

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...method

This protocol is a modification of the method of BIRNBOIM and DOLY[116]

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...medium

containing 50 40#40g/ml ampicillin.

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...primer

for data on the used primers, see table tab:seqprimers

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...step

The number of bases can be further changed by shortening or extending this step.

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...blue

The bromophenol blue serves as visible marker to check that the buffer gradient has formed correctly.

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...plates

To avoid producing air bubbles, the solution was poured in a continuous stream.

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...gel

Check for leaks between wells; dye should only be in alternate lanes and should not leak into adjacent lanes.

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...programme

Version 8.0-OpenVMS, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711, installed on OpenVMS AXP

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...gels

Its separation range is 100 to 1500 base pairs with a resolution down to 8 bp in the 100 to 200 base pair range.

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...mfold

mfold predicts optimal and suboptimal secondary structures for an RNA molecule using the most recent energy minimization method of Zuker[167, 168, 169, 170].

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