|PCR LABELING (labelling) AND AMPLIFICATION OF CLONED AND GENOMIC DNA SEQUENCES
Fast troubleshooting here. M13 primers here. Comments on primer design here
- 10x PCR buffer: Normally use a no Mg 2+ buffer', supplied together with Taq DNA polymerase, and containing 100 mM Tris-HCl, pH 8.3 and 500 mM KCl and sometimes detergents
- Unlabeled nucleotide mixture: 2 mM each of dATP, TTP, dCTP and dGTP in 100 mM Tris-HCl, pH 7.5 or water (Note 2)
- If you are labelling DNA, use Labeled nucleotide mixture: 1mM digoxigenin-11-dUTP (Roche), biotin 16 (or 11) dUTP (Sigma, Roche), fluorescein-dUTP or rhodamine-dUTP in 100 mM Tris-HCl, pH 7.5
- Primers: For labeling inserts in M13 and related plasmids with the same multiple cloning site (e.g. pUC18, pUC19 or pBluescript) use the universal M13 forward and reverse sequencing primers: 5'GTA AAA CGA CGG CCA GT 3' and 5'GGA AAC AGC TAT GAC CAT G 3' or variants (Note 1). The stock primer solutions (100 µM or uM) should be diluted to 10 µM
- 50mM MgCl 2
- Template DNA (Note 3)
- Taq DNA polymerase (1-5 unit/ µ l)
- Mix the following in a 0.5 ml microcentrifuge tube for PCR applications
|Y µ l
1.5 µ l
1.5 µ l
1.5 µ l
1.5 µ l
1.5 µ l
5 µ l
X µ l
49.5 µ l
Unlabeled nucleotide mixture
Labeled nucleotide mixture (Note 5)
Primer 1 (Note 1)
Primer 2 (Note 1)
MgCl 2 (Note 6)
10x PCR buffer
Template DNA (Note 3)
= Total volume
- Vortex and centrifuge to bring solution to the bottom of the tube, add 0.5 m l of Taq DNA polymerase to each reaction tube. Use a PCR machine with heated lid (old days: overlay with mineral oil, two drops per tube). Mix again by pipetting up and down, closing and sharply tapping the tube, and centrifuge to bring to bottom of tube and separate phases.
- Program your thermal cycler as follows
93 ° C 5 min
72 ° C 5 min
|94 ° C 30 seconds
56 ° C 30 seconds
72 ° C 90 seconds
|} 35 cycles
Hold 4 ° C
- Check the PCR product (labeling, size and concentration) by agarose gel electrophoresis
- Precipitate labeled PCR product with ethanol
- Primers can be synthesized to order or purchased: M13 universal sequencing primers, e.g. Sigma P4165 and P4290 or Stratagene #00303 and #300304.We use the M13 forward and reverse sequencing primers: 5'GTA AAA CGA CGG CCA GT 3' and 5'GGA AAC AGC TAT GAC CAT G 3'. (For some reason, they are much cheaper to order as custom primers than as products!) T7 and T3 sequencing primers are also suitable for many cloning vectors depending on the insertion site used for cloning. Many companies offer various primers to cater for more unusual insertion sites or other vector systems. Final primer concentration should be between 0.1 to 1.0 mM in the PCR reaction; 0.3 mM is suggested here. For designing your own primers, the program Primer3 is exceptionally good (find it with Google); we accept default parameters except usually ask for 1bp G/C clamp and 55% GC content.
- dNTPs: Store in 100 ul aliquots for 3 months at 20 °C. Some protocols recommend higher concentrations of nucleotides. Even pure nucleotides degrade over 12 months at 20 °C and should be replaced regularly. dNTP sets are available from e.g. Amersham, Roche, Sigma, York Bio. Stock solutions are either bought as mixtures (which give concentrations as the conc. of each nucleotide) or mixtures made up (reducing the concentration of each to 1/4 of the original). ("dTTP" in the sense of dCTP/dGTP/dATP does not exist - it is already deoxy, hence RNA has UTP. If you added dTTP it would be like adding a dideoxy nucleotide for the others, and stop further polymerase extension.)
- Clean uncut plasmid DNA from standard mini-preparations is diluted 1/50 to 1/1000 in water for use in the reaction. Typically, this amounts to 10 to 30 ng of template DNA (although a wide range, 10 pg to 100 ng, may be used).
- Many factors affect the results of PCR reactions: amount of template DNA, primer design, enzyme, conditions of amplification and magnesium ion concentration. No amplification or amplification of incorrect template sequences can occur if any is wrong. As the protocol given here describes the amplification of DNA sequence cloned in a plasmid where M13 primers are defined and the annealing at 55°C avoids polymerase pausing at unusual secondary structures of the insert DNA, optimization with multiple reactions and many different controls are not needed. The amount of template DNA is often difficult to estimate and it is advisable to set up test amplification of the desired size of DNA fragment without labeled nucleotide (if necessary adjust the amount of DNA template and other conditions) before repeating with the label in the reaction.
- Concentration of labeled nucleotide can be important as some haptens may reduce the activity of the enzyme. Possibly, use slightly more labeled nucleotide, especially with direct fluorophores. Biotin, being the smallest molecule of the widely used haptens, is normally accepted best by the enzyme.
- MgCl 2 concentration may need to be adjusted up or down; normally, a final concentration of 1 to 5 mM is suitable, being 0.5 to 2.5 mM above that of the total dNTP concentration.
- The M13 primers perform well at a high annealing temperature (55°C), and normally yield large amounts of a single PCR product. Other primers may need different annealing temperatures and more optimization.
- PCR amplification and labeling must be tested by gel electrophoresis for amount of amplification, product size and label incorporation (Figure 4.1, see Chapter 3 for methods, also see Note 3). Include a marker track with a known amount of DNA and compare the fluorescence brightness by eye after ethidium bromide staining to measure concentration and include a suitable size marker. When amplified by PCR (where probe length is defined by the distance between the primers), labeled probes run more slowly, because of the large labeled nucleotides, than control DNA amplified without label. It is useful to include control amplification without label to see the retardation as a measure of level of incorporation. If a fluorophore direct label has been used, the fluorescence of the labeled DNA fragment, and of the unincorporated nucleotides, will be visible in the appropriate color.
Revised October 2008.
Troubleshooting PCR - why my PCR does not work/fails - How to get PCR to work - No amplification in PCR
Our experience says three points are important:
1) Water - we routinely buy molecular biology grade water (eg. from Sigma). There are good reasons why molecular biology kits come with water: its quality is important!
2) Mixing. The reagents must be extremely well mixed. DNA solutions are viscous; enzyme is disolved in glycerol, so like your loading buffer it sinks. Mix tubes by pipetting up and down, then flicking the tube, then vortexing (unless you are using PCR amplification for fragments >5kb), then centrifuging and repeating. Particulalry for multiplex PCR reactions or multi-product reactions, mixing is a critical step.
3) The particular Taq polymerase make/manufacturer and associated buffer. If reactions don't work as expected (especially for amplification of multi-copy or difficult genomic sequences like IRAPs and SSRs, try a different Taq supplier.
Many books discuss aspects of PCR amplification. The New England Biolab website is useful - see link below. The Promega:website used to have useful information but as of 11/2007 this seems to have been deleted, along with their search engine and ways to access the site without logging in.
Useful details of methods reagents, dilutions and mixtures are given in the "Molecular Biology Techniques Manual" by Coyne et al. downloadable from http://www.mcb.uct.ac.za//pcrcond.htm The discussion "Taq DNA Polymerase Guidelines for PCR Optimization" http://www.neb.com/nebecomm/tech_reference/polymerases/taqdna_guidelines.asp is useful.