University of Georgia

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Parrott Lab

pMECA

A cloning vector with the the largest number of unique restriction sites around
Last modified November 20, 2003; information still current October 2004

What is pMECA?

pMECA is a cloning vector which contains 44 unique restriction sites, including 9 rare-cutters, all within the 230-bp polylinker. Traditional blue/white screening can be used to identify bacterial colonies with inserts cloned into the vector. Alternatively, bacterial colonies whose vectors contain inserts grow much faster than those without, permitting the size of the bacterial colonies to be used to determine those which contain inserts.

Strains that have been transformed with the vector:

  • DH5alpha JM109 HB101 Top10 (Invitrogen XL-1 blue (Stratagene)
  • DH5alpha (MCR)

Potential limitations:

  • Strain DH10B has been reported to not be transformable with pMECA. That appears to not be the case once pMECA has an insert. This can be an advantage in that only pMECA clones containing an insert grow well in this strain. We recommend transforming the vector into DH5alpha to grow up stock plasmid. Then this stock can be used to clone into and transformed into DH10B. In theory only the clones that appear on the plate should be recombinant.
  • The observation that XbaI digests pMECA poorly (if at all) can be explained by overlapping Dam methylation. The site is preceded by GA to give the sequence 5′-GATCTAGA-3′ (XbaI site underlined). The complementary strand is 5′-TCTAGA*TC-3′ (methylated A residue is marked with an asterisk, methylation sequence is in bold). NEB recommends growing the plasmid in strain GM2163 (catalog # 401-P) a Dam minus strain. They claim that it is an OK strain to use for plasmid preps even though it is endA1 positive.

Small colony size & slow growth

The phenotype expected from this vector is slow growth (small colonies). This growth characteristic is both on solid and in liquid media. Expect the cultures to take 24-48 hrs to appear on plates or to reach log phase in culture. However, this is the advantage of the vector. When the MCS is disrupted the growth of the cultures is normal (like that of pUC19).

Variable colony size– which to use

“We have transformed this plasmid [without an insert] into DH5 alpha and found two different sizes of colonies.  The majority of them are very very small and only a few of them are larger.  Which colony (very small one vs the bigger one) we should for the cloning experiments?”

Select the small colonies that appear on the plate and grow them up to make stocks. However, once the plasmid has an insert, select the large colonies.

Pointers

We would suggest for those first using the vector to plate the transformed cells on LB/Amp with IPTG and X-gal. Once you become familar with the growth characteristics, then remove the use of IPTG and X-gal. Bacteria transformed with the vector will also take longer to appear blue. One trick is to place the plates in the refrigerator (after growth at 37C) for several hours to better see the blue phenotype. Again, once you see how easy it is to pick with size, you won’t need to wait a long time for the small colonies to grow or become blue, just select the large colonies on the plate after 16 hrs of growth. We did not ever need to use chloramphenicol for the production of plasmid, but some may find it necessary.

How big is the difference in colony size?

What is the reference for pMECA?

  • Thomson, J.M., and W.A. Parrott. 1998. pMECA: A size-based, blue/white selection multiple common and rare-cutter general cloning and transcription vector. BioTechniques. 24:922-927. PDF.

Where can I find the sequence for the pMECA multiple cloning site?

  • Are there any restrictions on the use of pMECA?
  • pMECA is being made available on a non-exclusive basis for non-commercial research uses, with the following conditions:
    • pMECA will not be transferred to any third parties without permission from the originators of pMECA Any publications resulting from the use of pMECA will acknowledge the use of pMECA and cite the appropriate reference No guarantees, explicit or implied, on the effectiveness or efficiency of pMECA, are made by the originators of pMECA or The University of Georgia, nor can the originators of pMECA or The University of Georgia be held liable for failure of pMECA to perform as expected, or any consequences resulting therefrom
    • Anyone desiring to use pMECA for any commercial purpose must contact Shelley Fincher at The University of Georgia Technology Transfer Office for additional information.