Solutions

2.1.8(i) Denaturing Solution (0.5M NaOH, 1.5M NaCl)

20g of NaOH and 87.6g of NaCl were dissolved in 900ml of de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(ii) 50x Demhardt's Solution

lOg of Ficoll (type 400), lOg of polyvinylpyrrolidone and lOg of BSA (fraction V) were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water and stored at 4°C.

2.1.8(iii) 15% (w/v) Dextran Sulphate

150g of dextran sulphate was added to 800ml de-ionised water. The solution was heated to 65°C to dissolve the dextran sulphate. The solution was made up to 11 with de-ionised water and allowed to cool to room temperature.

2.1.8(iv) DNA Loading Buffer (0.25% (w/v) bromophenol blue, 0.25% (w/v)

xylene cyanol FF, 70% glycerol in TAB)

7ml of glycerol, 200p.l of 50xTAE (Section 2.1.8(xlvii)), 25ptg of bromophenol blue and 25/xg of xylene cyanol FF were mixed together. The solution was made up to 10ml with de-ionised water.

2,1.8(v) DNA Prep Solution 1 (50mM Glucose, 25mM Tris base, lOmM EDTA)

25ml of IM Tris base pH8.0 (Section 2.1.8(li)) and 20ml of 0.5M EDTA pH8.0 (Section 2.1.8(ix)) were mixed with 900ml de-ionised water. 4.5g of glucose was dissolved in this solution. The solution was made up to 11 with de-ionised water.

2.1.8(vi) DNA Prep Solution 2 (1 % (w/v) SDS, 0.2M NaOH)

0.8g o f NaOH was dissolved in 80ml de-ionised water. 10 ml of 10% (w/v) SDS (Section 2.1.8(xxxvi)) was added and the solution was made up to 100ml with de­ ionised water.

2.1.8(vii) DNA Prep Solution 3 (3M KAc in HAc)

300ml of 5M KAc (Section 2.1.8(xxv)) and 57.5ml of glacial acetic acid were added to 142.5ml of de-ionised water. The solution was well mixed.

2.1.8(viii) 50mM EDTA pHS.O

100ml of 0.5M EDTA pH8.0 (Section 2.1.8(ix)) was mixed with 900ml de-ionised water.

2.1.8(ix) 0.5M EDTA pHS.O

186.1 g o f EDTA was dissolved in 900ml de-ionised water. The solution was adjusted to pH8.0 with lOM NaOH. The solution was made up to 11 with de-ionised water.

2.1.8(x) 0.5M EG TA pH 8.0

190.2g of EGTA was dissolved in 900ml de-ionised water. The solution was adjusted to pH8.0 with lOM NaOH. The solution was made up to 11 with de-ionised water.

2.1.8(xi) ESP (0.5M EDTA pH8.0, 1% (w/v) lauroyl sarcosine, 2mg/ml

Proteinase K)

25fxg of lauroyl sarcosine and 5mg proteinase K were mixed with 2ml of 0.5M EDTA pH8.0 (Section 2.1.8(ix)). The solution was made up to 2.5ml with 0.5M EDTA pH8.0.

2.1.8(xii) Extraction Buffer (lOmM Tris-HCl pH8.0, O.IM EDTA pH8.0, 0.5%

(w/v)SDS)

10ml of IM Tris-HCl pH8.0 (Section 2.1.8(lv)), 200ml of IM EDTA pH8.0 (Section 2.1.8(ix)) and 25ml of 20% SDS (Section 2.1.8(xxxvii)) were mixed with 765ml de­ ionised water.

2.1.8(xiii) FISH Hybridisation Mix (50% (w/v) formamide and 1% (w/v) Tween 20

in IxSSC)

5ml of formamide, 4.9ml of 2xSSC (Section 2.1.8(xl)) and 0.1ml of Tween 20 were mixed together. Ig of dextran sulphate was dissolved in this solution, which was then sterilised by passing through a 22jLtm filter.

2 .1.8(xiv) FISH Solution 1

2.5/rl of 2mg/ml fluorescein avidin DCS and 2.5/rl o f 200/rg/ml rhodam ine-anti- digoxygenin were mixed with 1ml of 4STB (Section 2.1.8(xlv)).

2.1.8(xv) FISH Solution 2

10/xl o f 0.5m g/m l biotin-anti-avidin D was m ixed w ith 1ml o f 4STB (Section 2 .1,8(xlv)).

2.1.8(xvi) FISH Solution 3

2.5jLil o f 2mg/ml fluorescein avidin DCS. was mixed with 1ml of 4STB (Section 2 . 1.8(xlv)).

2,1.8(xvii) FISH Solution 4

5-6 drops of 2/rg/ml DAPI were mixed with 1ml of Citifluor.

2 .1.8(xviii) 50% {w/v) Formamide/2xSSC

50ml o f formamide, 40ml of de-ionised water and 10ml o f 20xSSC (Section 2.1.8(xli)) were mixed together.

2.1.8(xix) 70% (w/v) Formamide/2xSSC

70ml of formamide, 20ml of de-ionised water and 10ml o f 20xSSC (Section 2.1.8(xli)) were mixed together.

2.1.8(xx) 0.25M HCI

6.5ml of concentrated HCl (11.5M) was added to 293.5ml of de-ionised water and the solution was mixed.

2.1.8(xxi) 2M HEPES pH 6.6

476.6g o f HEPES was dissolved in 900ml o f de-ionised water. The solution was adjusted to pH6.6 and made up to 11 with de-ionised water.

2.1.8(xxii) Hexamer Mix (5x Oligo-labelling buffer; IM HEPES, 100/xM dGTP, dATP and dTTP, 250mM Tris base, 25mM MgCl^, 0.35% (w/v) P- Mercaptoethanol, 12.5U random hexanucleotide)

250/tl o f 2M HEPES pH6.6 (Section 2.1.8(xx)), 0.5/zl each o f 100/rM/ml dGTP, dATP and dTTP, 125^1 of IM Tris base pH 8.0 (Section 2.1.8(li)), 12.5/d o f IM MgCl^ (Section 2.1.8(xxvii)), 1.75/xl of p-M ercaptoethanol and 100/xl of 125 U/ml random hexanucleotide were mixed together. The solution was made up to 500/d with de­ ionised water and stored at -20°C.

2.1.8(xxiii) Hybridisation Mix

54ml of 15% dextran sulphate solution (Section 2.1.8(iii)), 8ml o f 50x Demhardt's Solution (Section 2.1.8(ii)), 12ml of 20xSSPE (Section 2.1.8(xlv)) and 4ml of 20% SDS solution (Section 2.1.8(xxxvii)) were mixed together. 2ml o f sonicated salmon sperm DNA was denatured by heating to 95°C for 5 minutes and then quenching on ice for 2 minutes and was added to the Hybridisation Mix after it had reached temperature in the hybridisation oven.

2.L8(xxiv) Hybridisation Wash 1 (2xSSC, 0.5% (w/v) SDS)

100ml of 20xSSC (Section 2.1.8(xli)) and 25ml of 20% SDS (Section 2.1.8(xxxvii)) were added to 875ml of de-ionised water and mixed.

2.1.8(xxv) Hybridisation Wash 2 (0.2xSSC, 0.5% (w/v) SDS, pre-warmed)

10ml of 20xSSC (Section 2.1.8(xli)) and 25ml of 20% SDS (Section 2.1.8(xxxvii)) were added to 965ml of de-ionised water and mixed.

2.1.8(xxvi) 5M KAc

490.7g of KAc was dissolved in 900ml of de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxvii) 0.075M KCI

5.59g of KCl was dissolved in 900ml of de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxviii) IM MgCl^

203.3g of MgClj was dissolved in 900ml of de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxix) 3M NaAc pH5.6

246.1 g of NaAc was dissolved in 900ml of de-ionised water. The solution was adjusted to pH5.6 and made up to 11 with de-ionised water.

2.1.8(xxx) 5M NaCl

292.2g o f NaCl was dissolved in 900ml of de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxi) 0 A M NaOH

16g of NaOH was dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxii) Neutralising Solution (0.5M Tris base, 1.5M NaCl)

60.6g of Tris base and 87.7g of NaCl were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxiii) lOM NH^OAc

770.8g o f NH4OAC was dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2 .1.8(xxxiv) 28% (w/v) PEG 8000/20mM

280g o f PEG 8000 and 4.1 g M gClj were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxv) 20% (w/yj PEG &000/2. TVaCZ

200g of PEG 8000 and 146.Ig of NaCl were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxvi) 30% (w/v) PEG 8000/1.5M NaCl

300g o f PEG 8000 and 87.7g of NaCl were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxvii) 10% (w/v) SDS

lOOg of SDS was dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xxxviii) 20% (w/v) SDS

200g of SDS was dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(ixl) SE Buffer (75mM NaCl, 25mM EDTA)

4.38g of NaCl and 9 .3 Ig of EDTA were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xl) IM Sorbitol/0. IM EDTA

182.2g of Sorbitol and 37.2g of EDTA were dissolved in 900ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xli) 2xSSC

100ml of 20xSSC (Section 2.1.8(xli)) was mixed with 900ml of de-ionised water.

2.1.8(xlii) lOxSSC

175.3g o f NaCl and 88.2g of sodium citrate were dissolved in 900ml of de-ionised water. The solution was adjusted to pHT.O with NaOH. The solution was made up to 11 with de-ionised water and sterilised by autoclaving.

2.1.8(xliii) OJxSSC/0.1% (w/v) SDS

5ml of 20xSSC (Section 2.1.8(xli)) and 5ml of 20% SDS (Section 2.1.8(xxxvii)) were mixed with 950ml de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(xliv) 20xSSPE (3M NaCl, 0.2M NaM^PO^.H^O, 0.02M EDTA)

175.3g o f NaCl, 27.6g of NaH2P04.H20 and 7.4g of EDTA were dissolved in 900ml de­ ionised water. The solution was adjusted to pH7.4 with NaOH. The solution was made up to 11 with de-ionised water and sterilised by autoclaving.

2.1.8(xlv) 4ST

100ml o f 20xSSC (Section 2.1.8(xli)) and 0.25ml of Tween 20 were mixed with 399.75ml of de-ionised water.

2.1.8(xlvi) 4STB (50ml 4ST, 1.5g BSA)

1.5g of BSA was dissolved in 50ml of 4ST (Section 2.1.8(xliv)).

2,1.8(xlvii) IxTAE

20ml of 50xTAE (Section 2.1.8(xlvii)) was mixed with 980ml of de-ionised water.

2.1.8(xlviii) SOxTAE (2M Tris-acetate, 0.05M EDTA)

242g of Tris base was dissolved in 800ml de-ionised water. 57.1ml of glacial acetic acid and 100ml of 0.5M EDTA (pH8.0) were added and the solution was made up to 11 with de-ionised water.

2.1.8(il) O.SxTBE

50ml of lOxTBE (Section 2.1.8(il)) was mixed with 950ml of de-ionised water.

2.1.8(1) lOxTBE (0.9M Tris-borate, 0.02M EDTA)

108g of Tris base and 55g of boric acid were dissolved in 900ml de-ionised water. 40ml of 0.5M EDTA (pH8.0) was added and the solution was made up to 11 with de­ ionised water.

2.1.8(h) TE (lOmM Tris, ImM EDTA)

10ml of IM Tris base (pH8.0) and 2ml of 0.5M EDTA (pH8.0) were added to 900ml of de-ionised water. The solution was made up to 11 with de-ionised water.

2.1.8(lii) IM Tris base pH 8.0

121.14g of Tris base was dissolved in 900ml de-ionised water. The solution was adjusted to pH8.0 with lOM HCl. The solution was made up to 11 with de-ionised water.

2.1.8(liii) 50mM Tris base pH8.0/20mM EDTA pH8.0

50ml o f IM Tris base pH8.0 (Section 2.1.8(h)) and 40ml of 0.5M EDTA pH8.0 (Section 2.1.8(ix)) were mixed with 910ml o f de-ionised water.

2.1.8(liv) IM Tris-HCl pH8.0

157.6g of Tris-HCl was dissolved in 900ml de-ionised water. The solution was adjusted to pH8.0. The solution was made up to 11 with de-ionised water.

2.2 Methods

2.2.1 Yeast DNA Purification

Y AC clones were obtained from Dr Steve Scherer (D epartm ent of Genetics, The Hospital for Sick Children, Toronto, Canada). The clones belong to the Hospital for Sick Children chromosome 7-specific (HSC7) library and are cloned in pYAC4 vector (Scherer, 1992).

The Y AC clone was plated on a YAC agar plate (Section 2.1.3(vi)) and incubated at 30°C for 48 hours. A single yeast colony of the YAC clone was picked from the plate and used to inoculate 15ml YAC Broth (Section 2.1.3(v)) in a 50ml tube, which was incubated for 48 hours at 30°C with shaking (200rpm). The tube was centrifuged at 900g for 5 minutes. The yeast pellet was resuspended in 500pl of IM Sorbitol/0.IM EDTA (Section 2.1.8(xl)) and transferred to a 1.5ml Eppendorf tube. lOpl of 50mg/ml zymolase 60,000 (Section 2.1.5) was added and the tube was incubated at 37°C for 60 minutes. The tube was centrifuged at 11600g for 1 minute. The pellet was resuspended in 500|uL1 of 50mM Tris pH8.0/20mM EDTA pH8.0 (Section 2.1.8(liii)). 50|il of 10%

SDS (Section 2.1.8(xxxvii)) was added and the tube was agitated vigourously, then incubated at 65°C for 30 minutes. 200pl of 5M KAc (Section 2.1.8(xxvi)) was added and mixed with the DNA solution and the tube was incubated on ice for 60 minutes. The tube was centrifuged at 11600g for 5 minutes. The supernatant containing the DNA was transferred to a fresh tube, mixed with an equal volume of isopropanol and the tube was incubated at room temperature for 5 minutes. The tube was centrifuged at 11600g for 1 minute, the supernatant was discarded and the DNA pellet was air-dried, then resuspended in 300pl TE (Section 2.1.8(h)). 15pl o f 1 mg/ml RNase A (Section 2.1.5) was added and the tube was incubated at 37°C for 30 minutes. 30pl of 3M NaAc pH5.6 (Section 2.1.8(xxix)) and 200pl of isopropanol were added and the tube was incubated at room temperature for 5 minutes. The tube was centrifuged at 11600g for 1

minute, the supernatant was discarded and the DNA pellet was air-dried. The pellet was resuspended in 100-300pl TE (Section 2.1.8(h)).

2.2.2 Preparation of Yeast DNA Plugs

YAC clones were obtained from Dr Steve Scherer (D epartm ent of Genetics, The Hospital for Sick Children, Toronto, Canada). The clones belong to the Hospital for Sick Children chromosome 7-specific (HSC7) library and are cloned in pYAC4 vector (Scherer, 1992).

The YAC clone was plated on a YAC agar plate (Section 2.1.3(vi)) and incubated at 30°C for 48 hours. A single yeast colony of the YAC clone was picked from the plate and used to inoculate 10ml YAC Broth (Section 2.1.3(v)) in a 50ml tube, which was incubated for 48 hours at 30°C with shaking (200rpm). The tube was centrifuged at 900g for 5 minutes and the yeast pellet was resuspended in 1ml of 0.5M EDTA pH8.0 (Section 2.1.8(ix)). The tube was again centrifuged at 900g for 5 minutes and this time the yeast pellet was resuspended in double the volume (of the pellet) of 50mM EDTA pH8.0 (Section 2.1.8(viii)) containing 1 mg/ml zymolase 60,000 (Section 2.1.5). The tube was incubated at room temperature for 10-30 minutes. An equal volume of 1.5% PBS LMP agarose (1.5% LMP agarose in PBS), pre-warmed at 38-40°C for 5 minutes, was added and mixed well. The mixture was then poured into 80/tl moulds ("plug formers" - supplied with PFGE equipment; Section 2.2.3) and allowed to set. The plugs were then blown out of the mould into a 15ml tube with a rubber pipette bulb. 5ml o f 0.5M EDTA pH8.0 (Section 2.1.8(ix)) containing 7.5% P-mercaptoethanol was added and the tube was incubated overnight at 37°C with gentle shaking (lOOrpm). The plugs were rinsed in 50mM EDTA pH8,0 (Section 2.1.8(viii)), then transferred to a 15ml tube containing 2.5ml of ESP (Section 2.1.8(xi)) and incubated at 55°C for 48 hours. The plugs were rinsed twice in 1ml TE (Section 2.1.8(h)) and then incubated in

1ml TE with 0.4mg/ml PMSF at 37°C for 2 hours. The plugs were rinsed well with TE and then stored in TE at 4°C.

2.2.3 YAC Pulsed-Field Gel Electrophoresis tPFGEl

PFGE to determine size of YAC clones was performed using the Pharmacia LKB 2015 Pulsaphor System. A 0.5xT B E /l% agarose gel was prepared using a gel former (supplied with PFGE equipment). Yeast DNA plugs (cut in half with a scalpel) were inserted into wells of gel and the wells were sealed with 1% LMP agarose using a glass pipette. Plugs of yeast chromosome DNA marker were inserted in the outermost wells and sealed in the same way. The gel was placed in the electrophoresis tank, which was filled with 0.5x TBE buffer (Section 2.1.8(xlix)) that had been cooled to 10°C, and the electrode array was put in place. The gel was run at 200V with a pulse time of 70 seconds for 15 hours and 120 seconds for 12 hours. Afterwards the gel was stained with 0.5/xg/ml ethidium bromide solution for 30 minutes and then destained with de­ ionised water for 30 minutes. A photograph of the gel was taken and the distance of marker bands from the wells was measured. The gel was then blotted (Section 2.2.8) and probed with radioactively-labelled total human DNA which had been purified from peripheral blood cells (Sections 2.2.6(i), 2.2.10(i) and 2.2.1 l(i)) in order to visualise the YAC clone. The size of the YAC clone was then calculated.

2.2.4 PAC Library Screening

Library filters of the De Jong Human Genomic PAC Library (in pCYPAC2N vector; unpublished) were obtained from the UK MRC Human Genome M apping Project Resource Centre (HGMP-RC) in Hinxton, Cambridgeshire. YAC DNA was labelled with a^^P-dCTP (Section 2.2.10(ii)) and hybridised to the PAC library filters (Section 2.2.11(ii)). Positive clones were then identified from the autoradiographs.