---+!!! Plasmid copy number determination Plasmid copy number is known to vary depending on origin of replication and culture conditions [refs]. Typically, plasmids are referred to in qualitative terms such as "High" "Low" "Single" copy yet the quantitative value can be altered with only slight/single bp changes in the origin of replication [refs] and dramatically alter both the per cell and population yield of other products coded on the plasmid [refs]. Here we describe 3 methods used in our lab and discuss advantages and disadvantages of each. %X% note, this page is in progress, and requires additional information . %TOC% ---++ Overview Regardless of what method of is used to evaluate the plasmid copy number, all methods are limited by the DNA extraction, and biological relevance of the sample when the DNA is extracted. To minimize bias in DNA extraction we recommend: never using a "plasmid prep" kit as these kits strongly favor isolation of plasmid DNA over genomic DNA, more? To maximize biological relevance we recommend isolating DNA under the same conditions as you are interested in or expect there to be a difference (stationary vs exponential growth; media choice; maintain antibiotic concentration; precondition cultures if reviving from frozen; do not grow/transfer more than required) ---+++ Comparison of methods in progress ---++ Agarose Gel Density 1 Day 1: Revive from freezer using small scrape of frozen culture to inoculate 5 mL of media with appropriate amount of antibiotic. Incubate overnight with appropriate shaking and temperature. 1 Day 2: Precondition culture by diluting 1:1,000 or 1:10,000 into 5-10 mL fresh media containing appropriate amount of antibiotic and culturing over night at the appropriate temperature/shaking conditions. This is important to remove effects of cryoprotectants. Recommend the use of saline intermediate if doing 10,000 fold dilution 1 Day 3: Inoculate up to 3 independent cultures from each preconditioned culture by diluting 1:100 or 1:1000. Fresh media and appropriate antibiotic should again be used, 24 hours of growth at appropriate temperature and shaking conditions. * *Alternatively*, cells can be cultured for less than 24 hours to measure differences in plasmid copy number in different cell phases. In that case, Day 4 work should be done on Day 3 as soon as cells enter appropriate phase 1 Day 4: Lyse cells and isolate DNA. 1 Centrifuge ~2x10^9 cells at 4000g for 5 minutes to generate cell pellet. 1 Carefully remove supernatant with suction/pipette. 1 resuspend cells by vortexing or agressive pipetting in 200 µl of lysis buffer. Make sure to fully resuspend. * lysis buffer: 200 µg/mL RNase, 2 mg/mL lysozyme, 10 mM Tris-HCL, 20 mM Na2EDTA. pH 8.0 * fresh? how should it be stored? 1 incubate at room temperature for 15 minutes 1 lyse bacteria by adding 200 µl of 2% SDS (in water), vortex throughly. 1 incubate in 65 C water bath for 30 minutes mix lightly at 15 minutes 1 Transfer 75 µl to new tube containing 25 µl of loading solution * post-lysis project expected to be slightly more viscus than water, and free of particulate/debris without need to centrifuge. * loading solution: 60% sucrose 0.25% bromophenol blue, 2% SDS, 20% ethanol. * add note about dye band location not being relevant to current work but could be removed/changed if influencing other projects. example image? 1 Store overnight at room temp in the dark. 1 Day 5: Gel electrophoresis. 1 load 20 µl of overnight lysate on 1% agarose gel (make sure all agarose is _completely_ dissolved * 8 well comb, 2 rows/gel, 40 mL volume, 2 uL SYBR safe (50% concentration), first lane ladder (control) * *Note:* gel must be made with TAE buffer which has very pure water (extra ions cause gel blurring). MillQ water works well 1 Run approximately 28 minutes on 130V in _fresh_ TAE buffer 1 capture image 1 only capture "1 gel" -- set intensity focus/adjustment for =intense bands= (as not to oversaturate these bands) 1 Day 6: data analysis 1 Fiji Batching samples -- Be sure to run each biological replicate on separate gel, unless number of samples so low as to fit all samples on single gel. -- When working with individual samples in other steps, order based on sample ID not based on replicate. Sample 1-12 replicate A sample 1-12 replicate B and so on. ---++ Next Generation Sequencing ---++ qPCR [[QPCRToQuantifyPlasmidCopyNumber][separate page describing method]] candidate for consolidation -- Main.DanielDeatherage - 11 Oct 2020
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Topic revision: r5 - 2020-10-12 - MattMcGuffie