Insert Purification, Plasmid Ligation and Transformation

PLASMIDS FOR SUBCLONING

• Add 1µg of plasmid (eg pBluescript KS+) to a microtube with a suitable buffer (eg One?Phor-All+) in volume of 19.5µl. Add 0.5µl of restriction enzyme(s) and incubate at 37°C for 2 hours.
  
  
• Add 180µl of water, 100µl of chloroform and 100µl of phenol. Vortex mix and spin for 2min at full speed.
  
  
• Collect the supernatant in a fresh microtube and add 200µl of chloroform. Vortex mix and spin for 2min at full speed.
  
  
• Collect the supernatant in a fresh microtube and add 20µl of 3M NaOAc, pH 7.0 and 500µl of ethanol. Mix and spin for 15 min. at full speed.
  
  
• Pour off the supernatant, pulse spin the residual liquid down and draw off with an autopippette, being careful not to disturb the (invisible) pellet. Allow the pellet to dry for 5min at room temp by leaving the microtube lid open.
  
  
• Resupend the pellet in 40µl of water. This is the cut plasmid at a nominal concentration of 20ng/µl. 1µl of this is usually sufficient for a ligation reaction.

Notes

It is not generally necessary to dephosphorylate cut plasmids for subcloning. However for library production it is essential to minimise plasmid religation and this method would not be applicable.
 
 

SUBCLONING RESTRICTION FRAGMENTS

• Digest DNA of interest to generate fragment(s) for subcloning, at a concentration which will allow easy detection of DNA in a 10µl volume after electrophoresis (100-300ng of fragment).
  
  
• At this stage the plasmid digests may be extracted, precipitated and combined directly in a ligation reaction or they may be purified from agarose gels. For gel purification, add 1.8g (for frags in range 750-5000bp, more agarose for lower fragment sizes) of agarose, 0.5ml of 50X TAE buffer and 26ml of water to a conical flask. Place a small conical flask, inverted, in the neck of the larger flask and put into the microwave for 1min at full power. Allow to cool, add 1.25µl of 10mg/ml ethidium bromide and pour onto a minigel tray (7x10cm). Insert the comb (use 0.5cm wide wells)and leave to set. Meanwhile make up (if not already in apparatus) 250ml of TAE with 12.5µl of 10mg/ml ethidium bromide. Add 2µl of 6X dye buffer to the restriction digest, assemble the gel in the apparatus, add the buffer if required and load 10µl of the sample along side suitable standards. Run at 60-80V until the bromophenol blue is about two thirds of the way down. Visualise the band(s) of interest on a UV transilluminator and excise in as small a gel volume as possible with a scalpel and weigh into a microtube.
  
  
• Add an equal volume of GFX gel extraction buffer (Pharmacia) and heat the gel slice 60°C for 15mins, mixing intermittantly. Add to assembled GFX extraction column and leave for 1min. Spin for 1.0 min at 10000g in the Jouan 4i. Wash the extraction column with 0.5ml of wash buffer by spinning through for 1.0 min at 10000g in the Jouan 4i. Place GFX extraction column in fresh microtube and add 50µ of water. Leave for 1min and spin for 1.5min as before.
  
  
• Add 5µl of 3M sodium acetate, pH7.0 and 150µl of ethanol to the eluate. Mix and spin at full speed for 15min to precipitate the purified DNA fragment. Wash with 200µl of 70% ethanol and allow to dry on the bench for 10 min.
  
  
• Add to the dry pellet 0.5-1.0µl (10-20ng) of suitably cut plasmid vector, 1µl of 10X ligation buffer (Helena), water to 9.5µl and 0.5µl of high concentration T4 DNA ligase (Helena). Mix and leave at room temperature for 4 to 16 hours.
  
  
• Proceed to copmpetent cell transformation and plating

Notes

Obviously gel purified inserts will result in plasmids bearing the required product. However sometimes it is desirable to ligate a mixture of restriction products, such as those from a recombinant l phage DNA digest, resulting in plasmids bearing a variety of restriction products. It is simple to digest such target materail with enzyme(s) of choice and then extract and precipitate the reaction mixture (as with the production of cut plasmids) without any gel purification step, and then use directly in ligation reaction as described above.

Plasmids and digests may also be double digested or sequentially digested and filled-in after digestion to generate partially filled or blunt ends.
 

Reagents

LB media; 10g tryptone, 5g yeast extract, 5g NaCl

LB agar; LB media with 10g/l agar

50X TAE buffer; 2M TRIS/Acetic acid, pH 8.0, 250mM sodium acetate, 50mM EDTA