---+!!! 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 method, 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 * Agarose Gel Density (AGD) is arguably the most easily accessible as it does not require large expense (NGS) or project specific items (qPCR primers), or uncommon expertise (both NGS and qPCR). * NGS and qPCR are much higher throughput, as they can both easily be done in plate format and with automated or semi automated analysis. AGD has an artistic flare requirement of getting good quality images, and using graphic user interfaces on a per sample/gel basis. * NGS has the advantage of not requiring additional work if you already have plans/reasons to sequence the sample (though it should again be stressed decisions such as how to extract DNA and when to extract it can have large implications) * qPCR allows semi automated computational analysis, but does require upfront design/test of plasmid specific primers that will likely vary from project to project. ---++ Appropriately Culture Cells The first 2 days of work, in getting cells to equilibrium are common to all analysis types. Additionally these 2 steps can be skipped if you are able to isolate cultures at biologically relevant times such as after transfers have been done in a serial transfer evolution experiment and do not need to revive cultures from -80° C glycerol stock. ---+++ Day 1: Reviving Strains * 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. * Biological replicates are recommended to be inoculated from separate ice scrapes at this stage. * %X% it is important to have a negative control that is the same strain without any plasmid grown in absence of antibiotic. ---+++ Day 2: Precondition Cultures * %X% This is important to remove effects of cryoprotectants which experience has shown can lead to oddities in the initial growth of the culture. * Dilute at least 1:100 into 5 mL fresh media again with appropriate amount of antibiotic * Incubate overnight with appropriate shaking and temperature. * *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 3 work below should be done as soon as cells enter appropriate phase. ---++ Agarose Gel Density <b>Lysis Buffer: </b> | *Reagent* | *Stock Conc.* | *Final Conc.* | *to 1 mL* | | Tris-HCl pH 8.0 | 1 M | 10 mM | 10 µL | | Na<sub>2</sub>EDTA pH 8.0 | 0.5 M | 20 mM | 40 µL | | ddH<sub>2</sub>O | - | - | 740 µL | | RNaseA | 20 mg/mL | 200 µg/mL | 10 µL | | Lysozyme | 10 mg/mL | 2 mg/mL | 200 µL | * Make lysozyme fresh from powder in ddH<sub>2</sub>O * Add enzymes last ---+++ Day 3: 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 Freeze cells in -80 C for at least an hour (that figure is a guess). Pellets can be stored for at least a couple months. 1 Remove pellets from -80 C and heat at 75 C in heating block for 5 minutes * Be careful pellet does not dry out. If the pellet dries, it can cause significant DNA degradation. 1 resuspend cells by vortexing or aggressive pipetting in 200 µl of lysis buffer. Make sure to fully resuspend. * lysis buffer: 200 µg/mL RNaseA, 2 mg/mL lysozyme, 10 mM Tris-HCL, 20 mM Na2EDTA. pH 8.0 * RNaseA and Lysozyme should be added fresh to lysis buffer on day of experiment as lysozyme is not stable for long term storage (ref). 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. Solution should be almost entirely clear at this step. 1 (Add 5 uL Proteinase K to each tube, vortex, and incubate 15 min RT -- still testing this step) 1 Spin at max centrifuge speed for 10 min in order to separate cellular debris 1 Remove ~350 µL of supernatant into new tube (optionally store at -80 C, or proceed to next step) ---+++ Day 4: Gel electrophoresis. 1 Transfer 20 µl of lysate to new tube containing 4 µl of 6x loading solution 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 * *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 post-stain gel * 15 µL GelRed + 50 mL TAE buffer * 30 min while gently rocking 1 destain with water, ~5 min gently rocking 1 capture image 1 proper light/filters for GelRed (same as EtBr) 1 only capture "1 gel" -- set intensity focus/adjustment for =intense bands= (as not to oversaturate these bands) ---+++ 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 ---+++ Day 3: DNA extraction ---+++ Day 4: Library prep ---+++ Day ?: Computational analysis ---++ qPCR This protocol is based on methods described in Lee _et al_ (2006), [[http://www.sciencedirect.com/science/article/pii/S0168165605007509][link to paper]]. ---+++ Day 0: Design and Verify primers before isolating DNA Design your primers as outlined [[http://barricklab.org/twiki/bin/view/Lab/QPCR][here]]. Once you have prepared genomic and plasmid DNA as below, verify by melt curve that your primers produce single products (typically a checkbox on the thermocycler software to run/report after the amplification steps). This can be done on any DNA containing both genomic and plasmid DNA. %X% note, genomic primers are often reused for same/similar strains. ---+++ Day 1& 2: see Appropriately Culture Cells above ---+++ Day 3 [[QPCRToQuantifyPlasmidCopyNumber][separate page describing method]] candidate for consolidation -- Main.DanielDeatherage - 11 Oct 2020
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Topic revision: r8 - 2021-03-30 - MattMcGuffie