Team:Brown/Notebook Protocols/movingdna

 Moving DNA (subcloning) 

Often pieces of DNA (biobricks, PCR fragments, promoters, etc) need to be moved into a different vector, combined with other pieces, or modified in various ways. This is usually accomplished as follows:

1) Restriction enzyme digest of plasmid and DNA pieces, making them compatible for recombination

2) Gel purification of each appropriate piece (agarose gel, excising appropriate DNA bands using Qiagen gel extraction kit – see manual fordetails)

3) Ligation of DNA fragment to vector

4) Transformation of new cells with ligation mixture.

Details of procedures:

1) Restriction enzyme (RE) digest: Each restriction enzyme works best in a particular buffer of pH, salt, etc etc. The REs are often compatible between different buffers, and the buffers are provided with the enzymes. Many websites give plenty of such information e.g. neb.com.

A normal digest is in 20μl as follows: 10x buffer - 2 μl Restriction Enzyme 1 μl (about 10 units) DNA (about a μg) Water to 20 μl (Remember the best order of addition) Incubate the digest reaction 1-2 hours at 37 degrees C, and run on a gel along with the plasmid digested similarly. A good stopping point is to freeze the digest after 1-2 hours at 37 degrees C, but do not let the incubation go longer. It is usually best if you can double digest with two different REs that will not only force the orientation of the cloning, but increase its efficiency. Compatible REs are best, but even if not possible, you simply isolate the DNA (like in the gel extraction procedure), or change the buffer after the first digest (depending on buffer components this may mean simply adding a salt, or changing the pH). If you know the sequence of your insert, make sure you check that the REs do not digest internally.

2) Gel purification procedure: Run a 1% agarose gel in TAE using gel loading buffer in your digest (so it stays in the well), and loading a ladder for reference. (See Qiagen kit manual for DNA purification)

3) Ligation of DNA fragment to vector: Following purification of DNA, mix isolated DNA such that copy number insert to vector is about 2:1 (remember staining intensity and size and mass considerations?) 	5x Ligation buffer 2 μl	(T4 DNA) Ligase 1 μl	DNA mixture XX μl Water to 10 μl Mix all components on ice (make sure ligase buffer was mixed thoroughly) with the pipette. Keep at 4 degrees C overnight. Before adding to transformation – dilute in water to 100 μl.

4) Bacterial cell transformation: This procedure is used to get DNA into bacteria. It requires fresh cells for optimal transformation – but it is quite reliable. Supercoiled DNA works much better than ligated DNA in transformations. the important part of this protocol is to plan ahead, and once cells are in CaCl2, to keep them ice-cold.

1. Grow a 5ml overnight culture of XL1-Blue in LB broth at 37 oC with vigorous shaking.

2. The next morning, add 50ul of this culture to a fresh tube of 5ml LB. Use the plastic 15ml snap top tubes. Again incubate at 37 o C with vigorous shaking, and grow to A600 = 0.6-0.7. This takes a couple of hours, and the suspension will begin to take on a silky appearance. Do not let this culture overgrow - the health of the cells declines with confluence. An alternative procedure is to start a culture of XL1-Blue cells from a recently streaked plate, (streaked on LB/tetracycline) and to grow for several hours.

3. Cool cells on ice for 10 min., then take the tops off the tubes and while using the green tube adapters, spin in Sorvall SS-34 rotor for 5 minutes, 4 o C, at 3,000 rpm.

4. Discard the supernatant and quickly put cells back on ice. [From here on, keep cells on ice as best you can, even when resuspending them in fresh buffers].

5. Resuspend the cells in 2mls of ice cold (sterile) CaCl2 (50mM) using your P-1000 Pipettman. Leave on ice for 15 minutes. (For 50 mM CaCl2 use 0.73 gms of CaCl2 - 2H2O per 100 mls of water, and autoclave)

6. Centrifuge the cells as in #3 above. Discard the supernatant and resuspend cells in 500 ul CaCl2. Use immediately for transformation or store at -70 oC. If cells used for transformation are frozen, remove tube from freezer and immediately put on ice. Allow these cells to thaw on ice, which takes about 30 minutes.

7. To transform competent cells, put 100-200ul of the suspension in a precooled 13 x 100mm disposable glass culture tube (microfuge tube works fine). Add DNA ligation mixture to the cells, mixing gently with your pipette on ice. [For a positive control of transformation, use 1 ul of supercoiled plasmid pBS DNA from any miniprep]. Cover the tube with parafilm and incubate for 30-40 minutes on ice. Prepare a beaker of water (tap water is fine) at 42 degrees C. Heat shock the cells by placing the glass culture tube at 42 degrees C for 2 minutes, and then back on ice. Add 0.5ml LB media and then incubate cells in the 37 degrees C water bath for 1 hour, making sure the tube is capped with Parafilm (or snapped for a microfuge tube).

8. Spread the cells over one or two LB/Amp plates, invert in 37 degrees C incubator overnight.