PCR cloning

You have been given some cDNAs (or if you know the DNA sequence and have the genomic DNA), and you wish to express the gene products for NMR or other biochemical/biophysical studies. First thing to do is to clone it into an expression vector, and the easiest way to do this is by PCR cloning. Decide first what kind of protein (i.e. fusion, native, tags etc.) you wish to produce. Then select your choice of vector (consider what kind of promoter, induction method, fusion partner, tags, etc. you want to use. There are large number of different expression vectors from different companies). For example, if you want to use a T7 promoter (which can give high level of expression and useful in cloning because there is no toxicity problem), you can use pET vector. Fusion to GST or other fusion partners are useful for protein purification and sometimes increases solubility (maltose binding protein is said to be the best for solubility) and stability of protein. Each system has its own merits and demerits, so consider carefully which one is best for the expression of your protein (although sometimes it may be more of a matter of trial and error). Once you've decided what to do, use the following procedure for PCR cloning.

1) DNA analysis

You should always do some preliminary analysis of your DNA (restriction enzyme analysis, codon usage, etc). There are quite a few web sites available for such analysis. You can also use GCG - login to chally first (UCL Biochem personnel only), then type gcg. The following programmes in GCG are useful: getseq (convert DNA sequence to gcg format), codonfrequency (check codon usage -look out for rare codons such as AGG, AGA, etc.), translate (translate DNA to protein), map (translate + restriction sites analysis), peptidesort (gives various data on your protein and cleavages by proteases - if you don't want long list of proteases cleavage pattern, just enter blank space when prompted) and many more. GCG user's guide and manual are available online.

2) Oligo Design

You begin by designing the oligos used to do the PCR - a well-designed oligo will get you off to a good start. See guide on primer design. You need the forward and reverse primers for the PCR.
For routine PCR reaction, it is not necessary for the oligos to be HPLC/FPLC, ion exchange or PAGE purified.

3) PCR

The forward and reverse primers are used to perform PCR. The PCR product can be purified using kits (Qiaquick, Geneclean etc.) or agarose gel electrophoresis (see step 5) and then digested with the appropriate enzymes. However, if the PCR product is to be ligated into TA cloning vector, go straight to step number 6 after purification. A brief note about TA cloning - this method involve cloning first into a cloning vector, then subcloning into an expression vector (i.e. digesting the cloned product then ligating into expression vector). TA cloning takes longer as it involve 2 cloning step but slightly easier. It is generally unnecessary to do TA cloning, however, if you are inexperienced, insecure or if you have problem digesting your PCR products, this may be the useful route to take. You need to use taq polymerase in PCR for TA cloning.

4) DNA digest

It is important that you make sure that the DNA is properly digested. For double digest, check by doing a separate digest reaction for each enzymes. Always used the appropriate buffer for the DNA digest, for example NdeI and XhoI may be digested at the same time (use Promega buffer D). But if different buffers are necessary for each enzymes, digest with one enzyme, then precipitate the DNA and digest with the second enzyme. See also NEB site and REBASE for more information on restriction enzymes.

5) Gel purification

It is essential before ligation that you gel-purify your digested or undigested PCR product as well as your cut vector. This is to remove your template DNA, unwanted digest fragments, enzymes as well as other contaminants. The DNA are separated by agarose gel electrophoresis in TAE buffer. Low-melting-point agarose is preferred. Cut out the desired DNA fragment (as small a fragment as you can manage without losing your DNA) and purify using Gene-clean or other methods such as freeze-squeeze, silica powders, etc. Or if you feel lazy, you can dispense with the purification step altogather (but only if you use LMP agarose for electrophoresis), just melt the agarose gel slices, dilute with water and add to ligation mixture (it works but expect lower efficiency).

6) Ligation

For directional cloning (i.e. ligation into 2 different restriction sites) and TA cloning, it is not necessary to dephosphorylate the vector DNA before ligation. You can leave the ligation mixture in fridge overnight, but if time is of the essence, incubate at 14-16 °C for an hour. A brief note on directional cloning: this is the preferred method as it obviate the need for phosphatase treatment and the desired product will be in the correct orientation.

7) Transformation

Use only super-competent cells for transformation. If you are doing blue/white selection, make sure that only the cells with the right genotype (lacZ Delta M15) such as DH5 alpha or JM109 is used.

8) Screening for recombinants

It is a good practice to always do a control ligation reaction with vector alone everytime you prepare cut vector. It is not necessary to screen for recombinant if there is very little background religation. However, if it is necessary to do so the are a number of protocols for screening recombinants.

9) DNA prep

Pick colonies from plate and inoculate into LB broth or any of your preferred media broth (miniprep 1-10 ml, midiprep 10-200 ml, maxiprep 200-2000 ml, gigaprep - you'd be mad even to think of doing this). Use kits if you wish - Qiagen, Hybaid, etc. Do an additional phenol-chloroform step if you wish to get clean DNA. Alternatively there are many cheap and cheerful protocols for DNA prep if you don't feel like wasting money on kits, e.g. alkaline lysis method, Merlin prep, boiling method, ammonium acetate method, CsCl method (take rather a long time but DNA clean), etc.(see also Molecular Biology protocols for some more DNA prep methods).

Finally... always sequence you DNA construct.

See Fermentas and ScienceXchange for other PCR cloning protocols. Other useful cloning protocol sites can be found at Michael Blaber's cloning PCR products, and at Carolina Workshop.

Originated by Chen,Last modified: Mon Apr 10 18:27:40 BST 2004
yue.zhang@unifr.ch