D N A Purification

2.1.2.3 Minipreps

A colony of E . c o l i was grown overnight in 4ml LB in a ro u n d b o tto m 15ml tube (F a lco n ) at 37°C w ith v ig o ro u s agitation. 1.5ml of the culture w ere pelleted at 13000rpm for 5min and resuspended in 100^1 of STET buffer (8% sucrose, 5% Triton X-100, 50mM Tris-HCl (pH8.0), 50mM EDTA). 10|il of a freshly m ade solution o f lysozym e (Sigma, lOmg/ml in lOmM

T ris-H C l (pH 8.0)) were added to the tube, w hich was then

boiled for 45s. Following centrifugation at 13000rpm for 15min, the pellet was rem oved with a toothpick and discarded. lOO^il o f ic e - c o ld i s o p r o p a n o l w e re a d d e d and th e tu b e w as centrifuged for 8min at 13000rpm. The DNA pellet was washed with 400)il of 80% ethanol, dried and resuspended in 50|il of TE ( p H 7 . 5 ) . 5 | i l w e re d ig e s te d an d a n a ly s e d by a g a r o s e

2.1.2.2 DNA purification using PEG

The follow ing p ro to co l was used for m aking D N A for sequencing. A 100ml o v ern ig h t cu ltu re was cen trifu g e d and cells resuspended in 5ml lOmM EDTA and left on ice for 5min. The cells were lysed with 10ml lysis solution (1% SDS, 0.2M NaOH) and 7.5ml of 3M Na Acetate (pH5.2) were added. After 5min on ice, the tubes were centrifuged at 4200rpm for lOmin and the supernatant was collected in a fresh tube. An equal vo lu m e o f ic e -c o ld iso p ro p a n o l w as ad d ed , the tube was inverted several times and left on dry ice for lOmin. DNA was collected by centrifugation at 4200rpm for lOmin. The DN A pellet was resuspended in 5ml of TE pH7.5 and an equal volume of PEG solution (20% PEG mwSOOO, 2.5M NaCl) was added, the tube was inverted several times and then left on ice for 5min. DNA was collected by centrifugation for lOmin at 4200rpm. The pellet was resuspended in 400pl of TE (pH7.5) and 2pl of RNAse so lu tio n ( 2 u / |il , S tratag en e) w ere added. T he so lu tio n was in c u b a te d at 37°C fo r I h , p r o te in s w e re r e m o v e d by P h e n o l / C h l o r o f o r m / I s o a m y l A l c o h o l ( 2 5 / 2 4 / 1 ) e x t r a c t i o n follow ed by C hloroform /Isoam yl alcohol (24/1). The D N A was precipitated using 0.1vol. 3M NaAc and 2vols. ethanol and the pellet rin sed with 70% ethanol before b eing re su sp e n d e d in 2 0 0 - 3 0 0 p l of TE (pH7.5). The quality and concentration o f the

DNA was assessed on a gel and by measuring optical density at

600 nm on a Beckman DU7400 instrument.

2.1.2.3 Caesium chloride gradient plasmid purification.

This m ethod was used to generate highly pure D N A for m ic ro in je ctio n . A 500m l o v e rn ig h t cu ltu re o f b a c te ria was centrifuged at 4000 rpm, the pellet was resu sp en d ed in 10ml resu sp e n sio n b u ffer (25m M T ris-H C l (pH 8.0), lOmM ED TA , 50mM glucose, lOmg/ml lysozyme) and left on ice for 15min. 20ml of lysis solution (as for PEG preps) were added follow ed by 15ml cold 3 M NaAc. The solution was gently inverted six times and left on ice for 10 min to allow for full lysis, then centrifuged at 4200rpm for 12 min. The supernatant was put in a fresh tube, the DNA was precipitated with 0.6 volumes o f cold

isopropanol and centrifuged at 4200rpm for 12 min. The pellet was resuspended in 10ml of lOx TE pH8.0 and the DNA was re­ precipitated with 10ml of 5M am m onium acetate and 20ml of ethanol. The pellet was resuspended in 3.9ml lOx TE pH8.0 and 4g CsC l were added together w ith 2 0 0 |il e th id iu m b ro m id e (lO m g/m l). This solution was tra n s fe rre d to a h e a t-se a la b le tube (Beckman) and centrifuged for 15h at 100,000 rpm in an u ltra ce n trifu g e (B eckm an).

The supercoiled plasmid D N A (lower band) was rem oved with a w id e -b o re syringe, d ilu te d to 4m l w ith w a te r and e th id iu m b ro m id e ex tra cted w ith 4x 8ml o f w a te r-sa tu ra te d iso b u tan o l. The D N A was p re c ip ita te d w ith 2 v o lu m e s o f

ethanol, resuspended in 500ml TE, then precipitated again with

40ml of 3M NaAc and 1ml ethanol (ice cold), and washed with 70% ethanol. The pellet was resuspended in 300 -5 0 0 p l o f water or TE (pH7.5) and the quality and concentration of the D N A was analysed by gel electrophoresis and optical density.