Plasmid Preparation

Label one 14 ml snap top and three 1.5 ml tubes for each prep

Store solution I and III in fridge

 

•  Centrifuge 5 ml bacteria culture 5 min @ 1500g

•  Dump broth, add 1ml LB, resuspend pellet and pour into 1.5 ml tube

……-   Pour ~60 ml LB into beaker, use serological pipet to quickly add 1ml LB to tubes

……- ..Cover tubes with kimwipe, vortex at angle to resuspend pellet

•  Centrifuge 1.5 ml tube 30 sec @ 12,000g, dump LB, quick spin, pipet off remaining LB

•  Resuspend bacterial pellet in 200µl ice-cold solution I, vortex to completely disperse

……- . Add to all tubes then vortex on tube rack to completely disperse

•  Add 400µl freshly prepared solution II, invert several times, do not vortex, store on ice

……-  Invert whole rack after adding solution II to all samples

•  Add 300µl ice-cold solution III, vortex (on setting 4) inverted for 10 seconds

•  Store tube on ice for 3-5 minutes

•  Centrifuge for 5 min @ 12,000g, transfer supernatant to a fresh tube

•  Add 500µl phenol:chloroform, vortex, centrifuge 2 min @ 12,000g

•  Transfer supernatant to fresh tube with 2 volumes 100% EtOH (or until tube is full)

…….- Use spray bottle to add ethanol

•  Store at room temp for 2 minutes

•  Centrifuge 5 min @ 12,000g

•  Dump EtOH, wash with 1 ml 75% EtOH

•  Centrifuge 1 min @ 12,000g, remove 75% EtOH with pipet

•  Air dry for ~10 minutes

•  Resuspend in 200 µl water or TE or EB

 

Solution I (500ml)………………………. Solution II (10ml) …………………………Solution III (40ml)

10ml .5M EDTA (pH 8)……………. 400µl 5N NaOH 24ml………………….. 5M Potassium Acetate

12.5ml 1M Tris-HCl (pH 8)……… 500µl 20% SDS 4.6ml…………………… Glacial Acetic Acid

4.5g Glucose …………………………………..9.1ml H₂O………………………………….. 11.4ml H₂ O

H 2 O (to 500ml)

Autoclave