ࡱ> ;=:O "bjbjCC .()e)ea T     !!!8Yu$!W$(#######$&(d#E #  $  ##Nz"G&L<Rj"#'$0W$r"+)B+)z"+) z"## W$+) : PicoGreen quantification of DNA or RNA Worth noting that most of the lab now use Qubit for low concentration DNA/RNA quantification, but PicoGreen is still good. For DNA: Ensure there is sufficient 1x TE, otherwise make more with the concentrated stock Dilute 20l  DNA stock (100g/ml) with 980l 1x TE to 2g/ml Keep this dilution in the bag for future use Work out a plate layout. You should do 2 replicates of each sample, and 2 replicates of the standard curve (5 wells x2 replicates). In a flat bottom 96-well plate, put 41 l TE in the 2 wells for the first dilution of the standard curve Put 50 l in the other wells for the standard curve (4 x2 replicates), and also in 2 wells for each sample you are testing Add 13.75 l of 2g/ml  DNA to the first well of one of the replicates of the standard curve, mix by pipetting Pipette 5.5 l out of the first well into the second, mix, pipette 5.5 l out of this well into the third, mix, pipette 5.5 l of this into the fourth well, mix and discard 5.5 l Repeat this process for the second standard curve replicate. Add 0.25 l DNA sample to be tested to each replicate well Dilute PicoGreen 1:200 with 1x TE (you need 500l for the standards, 100l per sample, plus 50l spare). Pipette 50 l PicoGreen dilution into each well (you can just put this on the side of the well without changing tip). Tap the plate a few times to mix Read the plate in a fluorimeter, excitation 488nm, emission 520nm, gain ~1000, but you can change this to bring different parts of the standard curve into range The concentrations of the DNA standards are: 500ng/ml, 50ng/ml, 5ng/ml, 0.5ng/ml Plot the standards and use Excel to calculate a formula for the best fit line, use this to calculate the concentration of the samples in ng/ml. For a dilution of 0.25 l DNA sample in 50 l, the concentration of the sample is 200 x the measured concentration For RNA: - kit R11490 life tech Note the sensitivity range of this assay for 0.2l RNA solution, this will measure concentrations between 500ng/l and 2ng/l. If your RNA concentration will be higher than this then dilute it (or just use the nanodrop). Ensure there is sufficient 1x TE, otherwise make more with the concentrated stock Dilute 4l ribosomal RNA standard stock (100g/ml) with 196l 1x TE to 2g/ml Spare aliquots of the rRNA standard are stored in the Illumina box in the -30 You can then make the dilutions directly in the wells of a 96 well plate. Set up two wells per dilution: Make dilutions: 1x TE 2g/ml RNA concentration 0l 50l 2000ng/ml 30l 20l 800ng/ml 45l 5l 200ng/ml 49l 1l 40ng/ml 50l 0l blank For each sample, do two replicate wells containing 50l 1xTE and 0.25l sample Dilute P &'(5U\n ( D l  : > IKL3;<Cû hL3hL3 h@GhL3hL3h2 h@Gh@Gh/mhSVth@GhZhYKhn5hn5hxom5hn5hn55 h@G5 hj6h@Gh@G6hcHh4'hcH5CJ(aJ(hgi5CJ(aJ(hn55CJ(aJ(4'(  j ?@23no 7$8$H$gdSVtgd(W gdcH$a$gdcHoOPrsghklm]^. 7$8$H$gd^ 7$8$H$gdn 7$8$H$gdSVt 7$8$H$gdL3CEFKh"#jklt klm^.9GR_gtuw !!~!%"&"9":"""""ǿǻǻǻÿǿǮǻǪ hSVth\`CJOJQJ^JaJh\`hgiU hSVth(hq=~h(hBxh^h8Dhmhn5 hYK5hYKhn55 hn5 hnhnhnhL3hIeIhSVt:.G_uw ~!!""""""""""""" 7$8$H$gdSVt 7$8$H$gdn 7$8$H$gdq=~ 7$8$H$gd^icoGreen 1:200 with 1x TE (you need 500l for the calibration curves, and another 100l per sample, plus 50l spare). So, for 6 samples dilute 5.75l PicoGreen in 1144l 1x TE Add 50l diluted PicoGreen to each sample Read the plate in a fluorimeter, excitation 500nm, emission 525nm, gain ~1000, but you can change this to bring different parts of the standard curve into range Plot the standards and use Excel to calculate a formula for the best fit line, use this to calculate the concentration of the samples in ng/ml. For a dilution of 0.25 l DNA sample in 50 l, the concentration of the sample is 200 x the measured concentration      FILENAME Pico Green Quantification v1.2 Houseley lab  PAGE 2 """"""""""""""""""""""""""̻ hSVth\`CJOJQJ^JaJh/m0JmHnHu hY0JjhY0JUhMhjhjmHnHuhYjhYUhjhU""""""""" 7$8$H$gdSVt,1h. A!"#$% w2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@6666_HmH nH sH tH @`@ nNormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. 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