Plasmid multimers as high resolution molecular weight standards for pulsed field gelelect rophoresis

Pulsed Field Gel Electrophoresis (1) makes postfble the separation and resolution of DMA BOlecules above the size range of constant ffeld agarose electrophoresfs. Currently, the size standards available for pulsed ffeld experiments consist of bacteriophage DHA oligomer "ladders" of various molecular weights (2). Here we describe a staple, rapid and reproducible method for construction of plasaid aultimers for use as standards in the 10-300 kb size range. We show the usefulness of such standards by sizing S*t II restriction fragments from a Streptococcus genome (ATCC #33748) using a ligated 10.1 kb £. coll plasmid, pZIp Neo (3). Fifty fig of plasmid DHA was digested to completion with BaoHl (New England Biolabs, Beverly, HA) at the unique plasmid BamH1 site. The linearized OKA was phenol extracted, ethanol precipitated and cast Into a 100 pi agarose "plug" as previously described (1). The "plug" was placed whole in 500 ml of ligation buffer (Bethesda Research Labs, Gaithersburg, MD), 100 units T4 DMA ligase (BRL) was added and ligatfon was allowed to proceed for two hours. 300 Ml ESP (O.SM EDTA, 10nM Trls, IX Sarcosine and 1 ag/ml Protelnase K) was added to halt the reaction and facilitate protein degradation. The plug was stored in this solution and 10 Ml "slivers" were removed and loaded as needed. Figure 1 shows a gel run at 9V/ca for 72 hours (15 second pulse time) on a pulsed ffeld apparatus constructed in this laboratory. Lane 3 contains the 10.1 Kb plasnid ladder loaded alongside a lambda phage concatemer ladder (2,4) in lane 2 and a whole Streptococcus genone digested with Sst II in lane 1. The genoaic restriction fragment sizes were determined through comparative densitoraetry of this and several gels and fragment sizes of 170,160,140,100,95,89,78,73,72,68,68,63,55,53,53,51,48,42,41,39,37,33,32,31,27,22,21,20,19,18,15 and 12 kilobases were found for a total genoae size of 1,845 kilobases. Ue note that the 10 Kb fragment of the plasmid ladder is usually circular and is