ࡱ> NQMO $bjbj3O3O .BQ%eQ%ejeHX]@8<%<taawhhh^``````$S! $dhhhhhh^h^ aPdJ0m$m$8m$6hhhhhhh~hhhhhhhm$hhhhhhhhh : Embedding cells in agarose Processing of cells while embedded in low meting point agarose provides the best possible preservation of high molecular weight DNA, while allowing enzymatic manipulation Plug preparation for mammalian cells Pre-heat waterbath to 50 Per plug, use 1x104 to 1x106 mammalian cells, freshly trypsinised (ethanol fixed cells not tested at time of writing), spin and re-suspend in 1ml cold L buffer Alternatively, 0.5-10mg frozen tissue can be used (we have only tested mouse liver) grind to a powder on liquid nitrogen and thaw directly in 1ml cold L buffer Cells can be stored for >1hr on ice in L buffer Spin cells1, re-suspend in 60l L Buffer per plug of 1x104 to 1x106 cells and transfer to 2ml tubes Melt an aliquot of CleanCut agarose by placing in a beaker with ~100ml water and microwaving ~1-2min Place tubes of cells at 50 for at least 2 min before adding agarose To make plugs, keep agarose at 50 (or in water it was boiled in). To 60l cell suspension, add 40l CleanCut agarose using a wide bore tip. Vortex for 5s, then pipette directly into a plug mould using a wide bore tip, 100l per plug Keep the plug mould on its side between plugs if cells are clumpy or you are using powdered tissue to avoid the cells sinking to the bottom of the plug Place plug mold on its side in fridge for ~30min to set Push plugs out of the mold into 2ml tubes containing 500l fresh digestion buffer, incubate at 50 overnight Skip to Plug washes section Plug preparation for yeast Pre-heat waterbath to 50 Per plug, use 1x106 to 1x108 yeast cells, either fresh but much more commonly fixed in 70% ethanol, spin 30s top speed and re-suspend in 1ml PFGE wash buffer Spin cells, re-suspend in 60l PFGE wash buffer per plug of 1x106 to 1x108 cells and transfer to 2ml tubes. Add 1l high grade lyticase solution per 60l. Melt an aliquot of CleanCut agarose by placing in a beaker with ~100ml water and microwaving ~1-2min Place tubes of cells at 50 for at least 2 min before adding agarose To make plugs, keep agarose at 50 (or in water it was boiled in). To 60l cell suspension, add 40l CleanCut agarose using a wide bore tip. Vortex for 5s, then pipette directly into a plug mold using a wide bore tip, 100l per plug Keep the plug mould on its side between plugs if cells are clumpy to avoid the cells sinking to the bottom of the plug Place plug mold on its side in fridge for ~30min to set Push plugs out of the mold into 2ml tubes containing 500l PFGE wash buffer containing 10l high grade lyticase solution and 1l beta-mercaptoethanol (14M) Incubate at 37 for ~2-3 hours Remove solution and replace with 0.5ml PK buffer, incubate at 50 overnight Plug washes Remove the proteinase K solution: Hold tube at a >45 angle, slide P1000 tip down top edge to bottom of tube before removing liquid. This ensures that the tip does not damage the plug they are VERY fragile Rinse the plugs with 1ml TE Wash plugs three times with 1xTE for >1 hour each with gentle rocking, include 1mM PMSF in washes 2 and 3 PMSF is from 100mM stock in ethanol, we normally add 1 drop of ~10l PMSF stock to each tube containing a plug after adding the TE. The concentration of the PMSF really doesnt matter much Remove the TE and add 200l TE containing 1l RNase T1 (Thermo ENO541) incubate 1 hour at 37 Remove the RNase solution and wash the plug with 1ml TE for 1 hour before storing at 4 in 1ml TE Store plugs for years at 4 in TE. The timings of all these steps are very flexible the PK incubation can be at least 4 days (we havent tried longer), the plugs can be left in any wash step for days without problem. Basic restriction digestion Cut plug in half and place in a 2ml tube with 100l 1x restriction buffer (normally CutSmart), incubate 30 min at room temperature We use a plug for all procedures, it is just easier. Replace the buffer with 100l fresh buffer containing 10-20U restriction enzyme, incubate the plugs 4 hours to overnight at 37 . Overnight at 50 is required for some enzymes which also works in our hands, increase reaction volume to 200l for these higher temperatures. Solutions L Buffer: 100mM EDTA pH8 10mM Tris pH 7.5 20mM NaCl Digestion buffer: 100mM EDTA pH8 10mM Tris pH 7.5 20mM NaCl 1% N-lauryl sarcosine 0.1mg/ml proteinase K Assemble this from stocks just before use, in the fume hood as the vapour from the 20% NLS stock is toxic PK buffer: 100mM EDTA 0.2% sodium deoxycholate 1% Na lauryl sarcosine 1mg/ml proteinase K Assemble this from stocks just before use, in the fume hood as the vapour from the 20% NLS stock is toxic 20mg/ml Proteinase K stock: 125l 1M Tris pH7.5 12.5l 1M CaCl2 781l 80% glycerol 11.58ml H2O 250mg Proteinase K (Roche) split into 1ml aliquots and store at -20( 1x TE 10mM Tris pH 7.5 1mM EDTA PMSF: 100mM stock in ethanol from Sigma PFGE wash buffer: 10mM Tris pH 7.6 50mM EDTA Lyticase stock: 17U/(l lyticase (Sigma >2000 U/mg L2524) 10mM KPi pH7 50% glycerol Store at -20( Note calculate lyticase stock using U/mg solid This grade of lyticase is insanely expensive so keep it solely for these preparations.  Speed and time will depend on the cell type. It is often best to do the cell washing and resuspension in a 15ml tube, but again depends on the cells  We buy this in 12ml bottles from Biorad and split into 6x2ml aliquots to minimise re-boiling cycles  Extend this time if you have cells for multiple plugs in a single tube the worst problems we see in plug making come from the cell suspension not being properly warmed up  You MUST use 2ml tubes plugs often fracture in 1.5ml tubes  For tougher samples, try using PK buffer instead, which contains 10x the amount of proteinase  Remember to use the low cell harvesting spin see our Harvesting Yeast protocol. Not doing this leads to low and inconsistent yields  We buy this in 12ml bottles from Biorad and split into 6x2ml aliquots to minimise re-boiling cycles  Extend this time if you have cells for multiple plugs in a single tube the worst problems we see in plug making come from the cell suspension not being properly warmed up  You MUST use 2ml tubes plugs often fracture in 1.5ml tubes  Hold tube at a >45 angle, slide P1000 tip down top edge to bottom of tube before removing liquid. This ensures that the tip does not damage the plug they are VERY fragile  You could use 1mg/ml RNase A for some applications we tend to avoid it due to its strong DNA binding activity. May not matter for a PFGE gel for example, but would in other cases  Always 2ml tubes!  NEB suggest doing this twice, which may be required for some enzymes  NEB have a table of which enzymes work well in plugs and whether they need extended (overnight) digestion  HYPERLINK "https://international.neb.com/tools-and-resources/usage-guidelines/digestion-of-agarose-embedded-dna-info-for-specific-enzymes" https://international.neb.com/tools-and-resources/usage-guidelines/digestion-of-agarose-embedded-dna-info-for-specific-enzymes     Embedding cells in agarose v2.1 Houseley lab  PAGE 3         # $ m ~   C E J d e t u y 鹲龭飾鹲龣²h~J, h,hyhyOJQJh)h)jhy0JU h$H* hyhy hyH*h$hyOJQJ^Jh: h)6h:h:6 hy6 hyh)hyhM5CJ(aJ(hy5CJ(aJ(5  L M } ~ I J {   L M ^gd$gdygd$gd) $a$gdyy Q R S o p z { | $ 0 > ? 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