Universal primers for amplification of cpn60 sequences
All of these primer sets amplify the "universal target" - the
to nucleotides 274-828 of the E.
sequence (not including
primer landing sites).
PCR reaction components and cycling conditions for cpn60 PCR from isolates are below. If you are looking for primers and protocols for cpn60-based microbiota analysis, see here.
Information and protocols related to PCR amplification of the type II chaperonin (thermosome) from Archaea are available here.
For additional information on the cpn60 universal target and its uses, visit cpnDB, the chaperonin sequence database.
Products from amplification with H279 and H280 are used in hybridization methods or can be cloned. To produce PCR products from pure templates for direct sequencing, H729 and H730 (which contain standard M13 sequencing primer landing sites) are used. An annealing temperature range of 46-50 C is a good starting place with these primers. (I=inosine, Y=C or T, R=G or A, K=G or T, S=G or C)
H729 (M13 sequencing primer underlined)
H730 (M13 sequencing primer underlined)
An unpublished alternative to H730 is H1134. This primer lacks the 3' TT dinucleotide of the H730 primer and can resolve occasional issues with "stuttering" in the reverse sequencing reaction that can occur in templates where the adjacent nucleotides in the amplicon are additional T's. The result of the string of T's from the primer and template is that sequencing results from the forward reaction are ideal, but the reverse sequencing reaction appears as two overlapping reads. H1134 is shown here with a T7 sequencing primer, but could easily be synthesized with the M13 reverse primer if desirable.
H1134 (T7 sequencing primer underlined)
A modification of the original universal primer amplification protocol is described in Hill et al. (2006), Environmental Microbiology, 8(4):741-746. The paper describes the application of a primer mixture containing a 1:3 molar ratio of primers H279/H280 and primers H1612/H1613 to improve amplification of G+C-rich templates from complex templates or individual templates. M13 sequencing primer landing sites can be added to the 5' end of these primers as in H729 and H730 to produce PCR products for direct sequencing.
An unpublished alternative to H1612 and H1613 are what we call the "Magic primers". These inosine-free primers offer an more economical alternative to other primers for amplification from G+C-rich templates. These primer sets work well on high G+C templates and have only showed difficulties with extremely low G+C templates (<40%). They also can be made with or without the M13 sequencing primer landing sites depending on your application. An annealing temperature of 57C is a good starting place with these primers.
H1594 (M1340F sequencing primer underlined)
H1595 (M1348R sequencing primer underlined)
PCR reaction components (Hill lab generic recipe for amplification of PCR products for direct sequencing with H729/H730 or H1594/H1595).
For a 50µL reaction:
|10X PCR buffer||1X|
|MgCl2||2 - 2.5 mM|
|Forward primer (H729 or H1594)||400 nM (20 pmol/reaction)|
|Reverse primer (H730 or H1595)||400 nM (20 pmol/reaction)|
|Taq polymerase||2.5 units/reaction|
|Water||to 50 µL|
We don't endorse any particular brand of Taq polymerase and have had success with many different types. Magnesium concentrations of 2-2.5 mM have proven the best in our lab).
Thermocycling parameters (we use an Eppendorf Mastercyler or a BioRad iCycler with 0.2mL thin-wall strip tubes and the instrument ramp rate set to approximately 2 degrees per second: 50% of maximum on the Eppendorf).
5 min at 94C
40 cycles of [30 sec at 94, 30 sec at annealing temp*, 45 sec at 72]
10 min at 72
*For H729/H730 our usual annealing temp is 50C. For H1594/H1595 we raise this to 57C. Some users report cleaner results using fewer cycles (24-30 instead of 40).
Purification and sequencing. Check the products of your reaction by loading 5µL on to a 1.5% agarose gel. Depending on the amount of primer-dimer and unincorporated primer you see, you may want to gel purify rather than just doing a PCR clean-up. We also have users who swear by the Amicon Ultra-0.5 30K column. Sequencing of purified products can be done with standard M13 sequencing primers as indicated on the primers above.